In order to explore the genetic diversity and genetic differentiation status of Schizothorax biddulphi, three different geographic populations of S. biddulphi in the Qarqan River, Kizil River, and Aksu Rivers were selected as the research objects. The mitochondrial DNA COII and ND4 gene sequences of 126 samples were compared and analyzed, and the differences and relationships between the two kinds of markers on the genetic diversity analysis results of S. biddulphi were discussed. The results showed that the A + T contents of the mitochondrial DNA COII and ND4 gene sequences of S. biddulphi were higher than the G + C contents, and the base composition was biased. Based on mitochondrial DNA COII and ND4 gene sequence analysis, 6 and 23 haplotypes were identified in 126 samples. In the mitochondrial DNA COII gene sequence, there were shared haplotypes in the three populations. In the mitochondrial DNA ND4 gene sequence, no shared haplotypes were found among the three populations. Based on the two kinds of markers, the haplotype diversity and nucleotide diversity of the three populations are 0.738±0.019/0.904±0.014 and 0.02129±0.00298/0.04876±0.00149, respectively. S. biddulphi is characterized by high haplotype polymorphism and high nucleotide polymorphism. The analysis of molecular variances (AMOVA) of the two kinds of markers showed that the genetic variation mainly came from the groups in all populations. Moreover, the genetic differentiation between the Qarqan River population and the other two populations reached a significant level (P <0.05). The BI tree constructed by Bayesian method (BI) is consistent with the haplotype network diagram, and S. biddulphi formed two branches. The mismatch distribution of the COII and ND4 gene sequences showed both bimodal pattern. This result indicates that the current distribution of S. biddulphi is the result of a second exchange between previously differentiated populations. The results also indicated that S. biddulphi had differentiated into 2 distinct geographic populations. In addition, when our research group studied the morphological difference analysis of different populations of S. biddulphi, it was found that the differentiation in some traditional morphological areas has reached the subspecies level. Therefore, it is suggested that the Qarqan River population should be managed as a separate population during the protection and development of germplasm resources.