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Currently, only three species are known in the genus of freshwater mussels Unio in European Russia, but no taxonomic validation by molecular data for these species yet exists. We sequenced partial COI genes for 23 individuals of Unio freshwater mussels, collected in the Volga basin from 3 regions located within an area of 550 km in latitude and 1200 km in longitude. Sequences obtained in this study were aligned with those taken from GenBank and analyzed using MrBayes and the MEGA7 programs. At phylogenetic trees, all sequences including obtained in this study form three major groups corresponding to three Unio species regardless of the methods of analysis. Sequences of geographically distant populations from West, Central and East Europe form homogenous species clusters supported by 100% bootstrap values on ML and MP trees and by 1.0 PP on Mrbayes tree. The levels of intra- and intergroup polymorphism for the COI gene estimated from genetic distances in Unio ranged from 0.001–0.004 and 0.036–0.152. Thus, intra– and interspecies genetic distances for the genus Unio did not overlap and are close to those obtained for other groups of mollusks. Therefore, populations of the genus Unio from the spacious European area are represented only by three species. These results did not support the existence of hidden species in the genus Unio.