Variation among complete cytb sequences (1140 bp) of Aphanius mesopotamicus Coad, 2009 was compared with closely related species, to investigate the validity of this taxon as a newly-described tooth-carp based on morphological characteristics. Maximum likelihood and Bayesian likelihood trees supported the monophyly of A. mesopotamicus and its sister group relationship to A. sophiae. Some 10-16 differences were found when compared to four different population samples of A. sophiae, whereas, intraspecific differences were only up to 6 bp. These distances suggest divergence from a common ancestor with A. sophiae at roughly 1 million years ago. These results are congruent with morphology-based hypotheses, indicating a recent speciation event.