Abstract: Prenant’s schizothoracin, Schizothorax prenanti (Tchang), a valuable and economic fish, distributes in the main streams and tributaries of the upper reaches of the Yangtze River in China. To assess genetic diversity and gene-tic structure of the wild populations of S. prenanti, we amplified partial Mitochondrial Cyt b gene sequences from 104 individuals of the four wild populations of S. pregnant (Wuxi, Chengkou, Ya’an and A’ba) from the upper reaches of the Yangtze River. The results showed that 43 polymorphic nucleotide sites and 25 haplotypes were detected among the Cyt b sequences of 104 individuals. The haplotype diversity was ranged from 0.704 to 0.884, and nucleotide diversity index was ranged from 0.007 to 0.012 among the four populations. Kimura 2-parameter distance was ranged from 0.008 and 0.017. The genetic distance between Ya’an and A’ba population was the smallest and the gene exchange between the two populations was frequent. The largest genetic distance was observed between Ya’an and Chengkou population, which might be explained by the little gene exchange between the two populations. AMOVA analysis showed that the genetic differentiation of S. prenanti was mainly occurred within populations variation. There were significant genetic differentiations between the groups, among populations within groups, and within populations. The results of neutrality test showed that the values of Tajima’s D and Fu’s F_s were not significant and the mismatch distribution was multimodal, indicating that the four populations of S. prenanti from the upper reaches of the Yangtze River had not experienced population expansion. This study provides valuable knowledge for protecting Schizothorax prenanti and theore-tic information for a rational utilization of the germplasm resources.