Flatfishes such as flounder and turbot are commercially important fish species. In this study, microsatellite loci were used to resolve the population structure of flatfishes collected across the Black Sea. Three mitochondrial genes, COI, 16S rRNA, and Cyt-b, were used for genetic identification and construction of phylogenetic trees. Available mtDNA sequences with known geographic information were retrieved from the GenBank database and included in the phylogenetic analysis to increase resolution. Pairwise comparisons of FST showed significant differences between surveyed sampling sites (P <0.01) and high genetic variability was detected across different geographic regions. Cluster analysis revealed the presence of three genetic clusters within the Black Sea. Significant genetic differentiation between Northern (Sea of Azov and Crimea) and Southern (Turkish Black Sea Coast) Black Sea sampling sites was detected. The Mantel test supported an isolation-by-distance model of population structure. These findings are vital for the long-term sustainable management of flatfishes and for the development of conservation programs.