Symbiotic nitrogen fixation enables legumes to supply most of their needs for nitrogen in efficient and ecological manner. Rhizobia are group of prokaryotic organisms with the unique ability to form symbiotic relationships with legume roots. In this relationship a new organ called nodule is formed, in which conversion of atmospheric nitrogen into ammonia, which is available to plants, occurs. Besides the significant quantities of nitrogen fixed in a symbiotic relationship, utilization of this process in legume production enables improvement of soil fertility and reduction of mineral fertilizer application costs. Bean (Phaseolus vulgaris L.) is very valuable and economically important culture. It is rich in proteins, fiber, potassium, phosphorus and B vitamin and recently, it has been discovered that bean consumption can contribute to health improvement. However, bean production in Croatia is limited to small-scale farmers and generally neglected despite favourable environmental conditions. Numerous rhizobial species can nodulate bean. Competitive indigenous strains of these bacteria which are well adapted to soil stress conditions can often be present in the soil. Their symbiotic efficiency is usually unknown. It is assumed that native rhizobial populations are also present in Croatian soils and that they contain strains resistant to stressful soil conditions and hidden potential for efficient nitrogen fixation. The aim of this study was to assess genotypic, phenotypic and symbiotic features of indigenous rhizobia isolated from soils of Northwestern Croatia. Soil samples were collected from 27 different locations in Northwestern region of Croatia. Trapping host method was performed to obtain 45 isolates of indigenous bean symbionts. The identification of isolates was first performed by Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) which is based on the characteristic protein profiles for each microorganism. Protein profiles obtained from isolates were compared to those contained in database created in Salamanca University, containing the type strains of all currently described species from the Family Rhizobiaceae. The results of identification by MALDI-TOF MS were further compared to those based on gene analysis. Random Amplified Polymorphic DNA (RAPD) method was employed to assess rhizobial genetic diversity and to choose representative strains for further analysys of gene sequences. Since the proposal for using the 16S rRNA (rrs) gene as universal tool for classification and identification of bacteria, this gene is the basis for rhizobial classification. Therefore, the sequence analysis of this gene was performed in this research to classify isolates obtained from bean nodules. In many studies it was shown that the analysis of the rrs gene is not sufficient for accurate differentiation among rhizobial species since several species with identical rrs gene sequences have been described untill now. These species are distinguishable on the basis of the housekeeping recA and atpD genes analysis, which are the most useful phylogenetic markers for the identification of rhizobial isolates. Sequence analysis of those two genes were further step in identification of rhizobia at species level. Bean is a legume nodulated by several fast growing species of family Rhizobiaceae, mainly belonging to genus Rhizobium. Some of those species contained several symbiovars being the symbiovar phaseoli most usually present. NodC gene is the commonly used phylogenetic marker to define symbiovars within species of genus Rhizobium and its sequences were analysed to study simbiovars within species of indigenous rhizobia isolated from Croatian soils. Phenotypic characterization of isolates included testing the tolerance to unfavorable soil conditions such as high levels of NaCl, different pH values and temperatures as well as intristic antibiotic resistance and assimilation of different carbon sources. Greenhouse experiment was setup using two factors (2 x 18) on the basis of a completely randomized block design with three replications. The first factor in the experiment were two traditional Croatian landraces of bean, Trešnjevac and Slavonski zeleni. The second factor were 15 indigenous rhizobial strains selected from different RAPD groups, reference strain used to inoculate plants, negative control (uninoculated plants) and positive control (uninoculated plants with addition of 0,05 % KNO3). At the flowering stage plants were collected and afterwords nodule dry mass, dry aerial biomass and total N in each plant were measured in order to estimate symbiotic efficiency of indigenous strains. Analysis of variance was performed with the statistical package SAS 9.4 for Windows. SAS Institute Inc. According to our hypothesis, indigenous rhizobial strains nodulating bean have been found in the soils of Northwestern Croatia. The results obtained with MALDI-TOF MS methodology showed that the strains from this study matched with different species from the Rhizobium leguminosarum phylogenetic group with score values higher than 2.0. Using this criterion investigated isolates were identified as Rhizobium leguminosarum, Rhizobium sophoriradicis, Rhizobium phaseoli, Rhizobium ecuadorense, Rhizobium acidisoli, Rhizobium indigoferae i Rhizobium pisi. The results of the RAPD analysis showed a high diversity among the isolated strains and the existence of 15 groups with similarity lower than 70 % from which representative strains were selected for core and symbiotic gene analyses. The rrs gene sequences were firstly compared to those available in Genbank and the results showed that all strains from this study belong to genus Rhizobium being their closest related species those of the phylogenetic group of R. leguminosarum. Similarity values of 100 % were found between 11 strains with R. leguminosarum and R. hidalgonense and between 1 strain (22BZ) with R. pisi. The rest of the strains, namely 8Z, 9T and 13T presented similarity values higher than 99 %, but lower than 100 % with R. leguminosarum, R. pisi and R. etli respectively. Nine out of 11 strains that were closely related to the R. leguminosarum phylogenetic group were identified as this species after the analysis of the atpD and recA housekeeping genes (similarity value higher than 98 %). Other two strains (25T and 26T) were identified as R. hidalgonense, a species recently isolated in America from P. vulgaris nodules. The strain 8Z formed an independent lineage more closely related to the species R. ecuadorense, but the similarity values were lower than 97 % in recA and atpD genes indicating that the strains represented by 8Z probably belong to a new species within genus Rhizobium. Analysis of recA and atpD gene sequence of strain 22BZ indicates that this strain belongs to the species R. pisi. Strains 9T and 13T formed an independent branch in the phylogenetic tree of housekeeping genes which was most closely related to R. sophoriradicis, but with similarity values lower than 98 %. Further research is needed to assign this strain to this species or to new phylogenetic lineage. The results of the nodC gene analysis of indigenous rhizobial strains isolated from Croatian soils showed that all of them belong to the symbiovar phaseoli which has previously been found in different Rhizobium species. Nevertheless we have found for the first time this symbiovar in the species R. pisi. Some differences were found between isolates regarding their phenotypic characteristic. Resistance to low pH is one of the most important traits of isolates since many Croatian soils are acid. Results of greenhouse experiment showed that both Croatian landraces were compatible with indigenous rhizobial strains. Significant differences were found between strains in their symbiotic performance. Nodule dry weight is parameter indicating nodulation capacity of strain. The highest nodule dry weight was obtained when Slavonski zeleni was inoculated with indigenous strain 26T (R. hidalgonense). No significant differences were found between this strain and strain 22AT (R. pisi). Symbiotic efficiency was estimated by comparison of dry aerial biomass of plants inoculated with indigenous rhizobial strains with dry aerial biomass of uninoculated plants. In this sence, the most efficient were strains 23T and 16T, both belonging to R. leguminosarum, species. The highest total N content was obtained in plants inoculated with strain 16T indicating this strain was most efficient in nitrogen fixation while similar values were obtained with strains 2Z, 23T i 1AT. Six out of 15 strains showed higher symbiotic fixation efficiency than positive control, while similar values for this parameter were obtained for five more strains. Least efficient were strains of species R. hidalgonense (20Z and 26T). Besides being highly efficient in symbiotic nitrogen fixation, strains 1AT and 2Z (R. leguminosarum) showed significant tolerance to unfavourable conditions such as high NaCl concentrations and high temperatures. Therefore, these strains should be included in further investigation in order to select high quality strain for application as biofertilizer for sustainable bean production. Given that the knowledge about the natural population of rhizobia that nodulate bean in Croatia is very limited, these study provided an insight into the composition and characteristic of this group of microorganisms.