Seven populations (Oľšava/Hornád R., Oľšava/Topľa R., Ublianka R., Ondava R., Ipeľ R., Malý Dunaj R. and Vlára R.) of Balcan spined loach were subjected to phylogenetic analysis based on variability of the cytochrome b gene and mitochondrial specific marker. They were separated in to five groups but these groups were represented by specimens from different populations. Genetic distances among populations and among groups were quite low. Subsequently, analysis of relations among our groups and sublineages of Danubian-Balkanian complex of Sabanejewia was made. the first four groups were genetically close to sublineage III, while the fifth one to the sublineage IV. These findings agree with the conclusion about conspecificity of analysed Danubian-Dniester populations belonged to monotypic S. balcanica. Phylogenetic analysis showed that the most suitable populations for the restoration of this species in the Bečva River are populations dwelling the Ipeľ and Vlára rivers.