The Burkholderia cepacia complex is a group of Gram-negative non-fermenting bacteria encompassing 24 closely related species known as native bacteria in environment but also as cause of infection in immunocompromised patients. Since there is currently no data of this complex in Croatia, the aim of this study was the identification and multilocus sequence typing of seven previously collected environmental isolates and one clinical isolate of Burkholderia cepacia complex in order to indicate the importance of knowledge on these rapidly spreading species influenced by human and animal waste. The analysis included 16S rRNA, lepA, atpD, gyrB, gltB, phaC, recA and trpB genes. Predetermined identification of potential Burkholderia spp. isolates was confirmed by molecular identification and phylogenetic analysis of 16S rRNA gene. The sequence types of isolates were determined by multilocus sequence typing. The results of the analysis revealed that isolates belong to B. multivorans, B. cenocepacia and B. ambifaria species. Moreover, new alleles (genotypes) and sequence types (ST) of these species were recorded. The species of Burkholderia cepacia complex are usually multiresistant to antibiotics and possible resistance mechanisms of these isolates by analysis of blaTEM gene encoding ß-lactamases were performed. Early identification of the Burkholderia cepacia complex species is extremely important in preventing the transmission of infection caused by these bacteria and the correct choice of antibiotics in their treatment.