The main goal of this thesis was to determine and analyze the diversity and genetic structure of populations of Alburnus sarmaticus (Pontian shemaya) in Croatia. In Europe, this species lives only in the Black Sea basin, in the downstream parts of the Danube, and in the Dnieper and Southern Bug in Romania, Ukraine and Bulgaria, while in Croatia a completely isolated population of this species can be found in the Sava, Kupa and Mrežnica Rivers. Samples were collected from the Mrežnica and Kupa Rivers. Data analysis includes isolation of total genomic DNA, amplification of selected gene markers by polymerase chain reaction, sequence alignment, which I performed using the BioEdit program, during which individual mutations between sequences were observed. I also made phylogenetic trees using the PAUP program, using the methods of the highest parsimony and the highest probability. I determined that the sequences of the Pontian shemaya belong to the monophyletic line. I then constructed a phylogenetic network of the obtained haplotypes using the Network program. I used the DnaSP program to determine the degree of intrapopulation gene diversity. I analyzed the intraspecific and interpopulation structure of the Pontian shemaya in Croatia in detail using the MIGRATE program. Phylogenetic trees show that the species A. sarmaticus belongs to the old monophyletic line. The results also showed high genetic diversity, which is a very positive indicator for the future favorable development of these species.