Squalidus argentatus is a common freshwater fish distributed in Yangtze River, including in the main and tributary river. In this study, the genetic diversity and structure of S. argentatus from the upper and middle Yangtze River were analyzed using mitochondrial DNA Cyt b gene sequence. A total of 217 individuals were collected from 7 sampled sites, including Jiangjin, Jingzhou, Jianli, Honghu and Huangshi in Yangtze River, and Chishui in Chishui River and Yingtian in Xiangjiang River, the two tributaries of Yangtze River. A total of 81 polymorphic sites and 97 haplotypes were detected and the average haplotype diversity (Hd) and average nucleotide diversity (Pi) were 0.966 and 0.008 6, respectively (Table 1). Analysis of molecular variance (AMOVA) suggested that significant genetic differentiation occurred between the Chishui population and the others, and no genetic divergences among the latter six populations were found by pairwise AMOVA analysis (Table 3). The neutral test showed that historical population expansion did not occur in S. argentatus (Fig. 3).