Members of Nemacheilidae Family, Turcinoemacheilus genus were subjected to molecular phylogenetic analysis in this study. This genus was reported in 2009 to inhabit in Karoon River drainage, in contrary to previous assumption that it was the endemic species in the Basin of Tigris River. It was sampled from three stations placed in different tributaries in Karoon drainage and evaluated to understand the molecular phylogenetic relationships of Turcinoemacheilus in Iran. The mitochondrial cytochrome b gene (cyt b) and control region were used to infer phylogenetic relationships. PCR amplification of control region was not carried out successfully, possibly due to the high divergence of this sequence in the studied genus. The amplified fragments of cyt b were sequenced then analyzed by the use of phylogenetic software. Only one divergent position was seen in all three samples stations located in amino acid position 365. GTR and p-distances of cytochrome b gene for T. kosswigi computed from different stations of running water in Karoon drainage showed these samples belong to different populations and fall in intraspecific differences. In this study, examination of the molecular phylogeny using Bayesian analysis, Maximum Parsimony or neighbor-joining define the phylogentics of Turcinoemacheilus genus as a monophyletic clade which is sister-clade of Nemacheilus and Schistura genera. This report is the first report of Turcinoemacheilus molecular data and could describe molecular phylogeny of this genus in loaches.