Taxonomic investigations of 4 species of the genus Cobitis: C. simplicispina Hanko 1925, C. taenia Linnaeus 1758, C. albicoloris Chichkoff, 1932 (= C. peshevi Sivkov & Dobrovolov, 1984) and C. elongata (Heckel & Kner 1858) from Turkey and Bulgarian freshwaters were carried out using biochemical-genetic methods. The analyses of one general muscle protein (PROT) and six enzyme systems including esterase (EST), lactate dehydrogenase (LDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), malic enzyme (MEP), malate dehydrogenase (MDH) and superoxide dismutase (SOD) revealed electrophoretic differences between the species examined. Fractions controlled by the LDH-B locus of C. simplicispina gave completely different electrophoretic mobility from those of the other Cobitis species. Nei's genetic distance (D-Nei) between C. simplicispina and C. albicoloris, C. taenia and C. elongata was estimated as 1.39, 1.16 and 1.27, respectively. C. simplicispina might have diverged from the other three species 5.8 - 6.95 million years ago.