Abstract: Fish population genetics have mainly focused on economic fish or endangered species, while the knowledge on genetic structure of the fish with low economic value is limited. In the present study, we collected a total of 323 sharpbelly (Hemiculter leucisculus) individuals from 13 populations in Yangtze River Basin and the affiliated lakes to evaluate their genetic diversity and population structure by amplifying the mitochondrial DNA Cyt b gene fragments (1100 bp). Genetic diversity analysis showed the pattern of high haplotype diversity and high nucleotide diversity, suggesting that the populations in the Yangtze River Basin were fairly stable. In addition, based on mitochondrial cytochrome b gene phylogenetic analysis, there were five mitochondrial lineages (lineage A-F). The neutral test of Fu'Fs and mismatch distributions with negative Tajima's D values indicated that the lineage A, B, E, and F underwent population expansion, and the expansion was from the upper section to the middle section of the Yangtze drainage. Fst and phylogenetic analysis and the significant divergence among lineages A-F implied that there are at least four different species on the level of mitochondrial DNA in the Yangtze River Basin. The population genetic structure and diversity of Hemiculter leucisculus may reflect the distribution pattern in Yangtze River Basin.