Genetic diversity of three pure populations of Cobitis elongatoides from hydrologically and geographically different drainage areas, the Lužnice River (48.49.60 N, 14.55.60 E, Labe River drainage area, North Sea basin), the Nová Říše reservoir (49.09.11 N, 15.32.464 E, Morava River drainage area, Black Sea basin), and the Ida River (48.32.284 N, 21.03.421 E, Tisza River drainage area, Black Sea basin) was evaluated by sequencing of mitochondrial cytochrome b gene. Species identification of unknown Cobitis samples from Vipava River drainage area in Slovenia was also performed based on cytochrome b sequence. All forms of the genus Cobitis are protected by national legislatives and, in accordance with the Council Directive No. 925/43/EEC, they enjoy protection in selected localities of European importance. In total, 11 haplotypes of Cobitis elongatoides were found in Czech and Slovak waters, manifesting low overall genetic diversity. Samples from Vipava River drainage area were identified as Cobitis bilineata.