Abstract: The rock carp, Procypris rabaudi (Tchang), is an endemic and economic fish in the upper reaches of the Yangtze River and its biological resource had markedly declined. This study analyzed mitochondrial DNA (mtDNA) cytochrome b (Cyt b) gene sequence analysis to investigate the genetic diversity of 161 individuals in Chishui ranges and Wanzhou ranges of the upper Yangtze River from 2013 to 2017. The results indicated that the average content of base composition was 30.2% A, 27.9% T, 28.2% C and 13.7% G, and the content of A+T (58.1%) was significantly higher than that of G+C (41.9%). The data showed a strong bias against G. Fifteen haplotypes were identified in 161 sequences, and 15 haplotypes were defined. The overall haplotype diversity index (H_d) and nucleotide diversity index (P_i) were 0.590 and 0.00132, respectively. The results of phylogenetic tree by Maximum likelihood method were consistent with haplotype network map, and no obvious geographical distribution pattern was formed. The genetic distance between the two populations was 0.001, and the mean genetic distance within the population was 0.001. F_st statistics showed that the F_st (fixation index) value was 0.01749 (P>0.05), and there was no significant partition differentiation between the two geographical populations. There were frequent gene flows between Wanzhou and Chishui populations (value of Nm is 24.71). Analysis of Molecular Variation (AMOVA) on Cyt b gene revealed that the largest component of variance mainly occurred within populations (98.25%). The neutrality test indicated that the population expansion had already occurred among the rock carp in Wanzhou populations about 0.15 million years ago. According to the comprehensive analysis, the genetic diversity of Chishui population is lower than that of Wanzhou population, but there are widely gene flows among these two local populations. The genetic diversity of rock carp is relatively lower than other endemic fishes. It is urgent to propose reasonable management measures to strengthen the protection of this species in the Yangtze River.