The Mesopotamian spiny eel Mastacembelus mastacembelus is an endemic freshwater fish confined to the Euphrates and Tigris drainages of Mesopotamia. In this study, it was aimed to reveal the genetic variation of Mesopotamian spiny eel in Turkey with samples from the distribution regions in the Tigris and Euphrates river basins. For this purpose, the partial mitochondrial COI gene region (656 bp) was sequenced from 15 samples from seven populations (Aksu, Merzimen, Karasu, Sinnep, Ambar, Zarova streams and main body of Tigris River). Eight variable nucleotide positions were identified and three of which were polymorphic. Overall haplotype and nucleotide diversity are Hd= 0.642±0.081 and π=0.00339±0.00069. Four haplotypes were identified, one from the Tigris River basin, and three from the Euphrates River basin. Haplotype network analysis contains four unique haplotypes with at least one mutational step and no haplotype shared between main river drainages. The most common haplotype was H1 and three Tigris populations shared it. The phylogenetic inferences suggest that Mastacembelus populations are divided into two main clades. The first clade consists of haplotypes of the Tigris River basin while the second clade contains Euphrates River basin populations.