Abstract: Schizothoracinae fishes that belong to the family Cyprindae are a group of endemic species living in the Asia Plateau. Schizothorax biddulphi is a unique Schizothoracinae fish of the Tarim River system. It is included in China Red Data Book of Endangered Animals and second-grade protected animals of Xinjiang Uygur Autonomous Region. To investigate the genetic structure and genetic diversity of S. biddulphi, five populations of S. biddulphi were collected. Through amplifying the first hypervariable segment of mtDNA control region, 95 sequences with length was 456 bp were acquired. Eight variable sites were detected, six of which were parsim-info sites, and two of which were singleton sites. All eight nucleotide substitutions were transitions. The contents of cytosine, thymine, adenine and guanine were 17.15%, 31.53%, 36.33%, and 14.99%, respectively. There were ten haplotypes in 95 sequences. Haplotype diversity and nucleotide diversity were 0.7118 and 0.0024, respectively. The genetic diversity of the Tashikuergan population was the highest among these five populations. The result of AMOVA showed significant difference among S. biddulphi populations, 44.27% molecular variation was among populations, and 55.73% molecular variation was within populations. The pairwise fixation index (FST) revealed significant difference among all S. biddulphi populations except between the Kalakashi River population and the Yulongkashi River population. Neighbor-joining tree was constructed using two Barbinae fishes as outgroups. The bootstrap values were low at the interior branch points of S. biddulphi. Median-joining network of S. biddulphi showed a star-shaped structure. There was a main haplotype in the network, and most of the other haplotypes were separated by one-step mutations from the main haplotype. Two-step and three-step mutations were observed in Tashikuergan population only. Fu’s Fs test resulted in negative values and was statistically significant. Mismatch distribution of S. biddulphi appeared to be unimodal, which closely matched the expected distributions under the sudden expansion model. The observed value of the age expansion parameter (t) was 1.1680 and the time since population expansion was estimated to be 0.2 million years ago. The movement of Tianshan and Kunlun Mountain, the Quaternary interglacial-glacial cycles might have affected the demographic history of S. biddulphi.