Cobitis is a Palearctic genus of ray-finned fish in the Cobitidae family, also known as spined loaches. The classification within Cobitidae family is still disputed, the number of recognized species varies according to the number of morphological traits considered. With the development of the molecular markers and methods, within the Cobitis genus, more than 80 species were identified. The most common species for Danube and Dniester River Basins are Cobitis taenia, Cobitis tanaitica, Cobitis elongatoides and belongs to the Cobitis taenia hybrid complex. These species have a particular type of reproduction (gynogenesis) that leads to an asexual lineage (diploid, triploid and rarely tetraploid individuals). Cobitis elongatoides was exclusively the maternal ancestor of all the C. elongatoides-tanaitica hybrids, while the hybridization process was reciprocal within the C. elongatoides-taenia complex. Cobitis elongatoides, commonly named spined loach, is native to Danube basin, upper Elbe and Odra drainages, and also Dniester River. The aim of this study is to highlight the variability and phylogenetic relationships between the Cobitis elongatoides individuals from different river basins analyzing the molecular data provided by the mitochondrial marker cytochrome b gene. Total DNA extraction for the captured individuals from Prut and Dniester Rivers was performed using phenol chloroform isoamyl alcohol protocol. In order to amplify the cytochrome b gene, the L15267 and H15891 primers were used. The amplicons were successfully sequenced using CEQ 8000 Genetic Analysis System (Beckman Coulter). Genetic diversity within and between hydrological basins was assessed in Arlequin (v 3.5) software, computing general molecular indices. Our data reveal a high similarity between C. elongatoides individuals and C. elongatorides x C. tanaitica hybrids that belong to different tributaries of the Danube river basin, pointing that C. elongatoides is the maternal ancestor of all C. elongatorides x C. tanaitica hybrids.