The Chinese Catfish Silurus asotus is a commercially valuable aquaculture fish in some regions of China and Japan, and it is widely distributed throughout the freshwater reservoirs, lakes and rivers of China. Due to overfishing, environmental pollution, water conservancy, human production activities and other causes, wild Chinese Catfish has declined rapidly. Its fishery resource has been gradually exhausted in the Huaihe River Basin in the past decades. In order to analyze the genetic structure and demographic history of S. asotus, the mitochondrial cytochrome b(Cyt b) gene sequences were used in the study. The results showed that Cyt b gene sequence contained 841 bp nucleotides and the T, C, A and G contents were 28.59%, 29.55%, 27.44% and 14.42%, respectively. A total of 40 nucleotide sites and 32 haplotypes were obtained from 121 individuals collected in 7 different regions, including Xinyang, Huaibin, Bengbu, Hongze Lake, Yinghe River, Pihe River and Chihe River. The average haplotype diversity (h) and nucleotide diversity (P_i) were 0.884 8, 0.003 8, respectively, showing that there was a high level genetic diversity in the populations ofS.asotus from Huaihe River basin. The AMOVA analysis (total F_st=0.115 0) and the level of N_m values (3.58), as well as the average K2-P distances (0.002-0.009) all revealed that the S.asotus had no obviously geographical differentiation. However, the phylogenetic tree estimated by neighbor-joining (NJ) method indicated that the individuals of S. asotus formed two monophyletic lineages, and the two lineages were not correlated with geographical populations. Neutrality tests, mismatch distribution and network analysis suggested that this species underwent population expansion in the middle and late phase of middle Pleistocene following bottlenecks and/or they originated from a small number of founding individuals. The time that the total population of S. asotus expanded in the Huaihe River was estimated to be 0.17-0.29 million years ago.