Abstract: To explore genetic structure of 164 Bombay duck (Harpadon nehereus), fishes from Qingdao, Nantong, Zhoushan, Sanmen, Ningde, Quanzhou and Zhanjiang in the coastal areas of the Yellow Sea, East China Sea and South China Sea were collected to sequence the mitochondrial DNA cytochrome b (Cyt b) gene fragment. Thirty-two mutation sites, including 27 single mutation sites and 1 simple mutation site were detected. The 164 sequences defined a total of 29 haplotypes. The average haplotype diversity (H_d) and nucleotide diversity (π) were (0.3026±0.0479) and (0.000371±0.000379), respectively, and both the lowest haplotype diversity and the lowest nucleotide diversity were Quanzhou population. The average genetic distance among different populations was 0.00035 and the genetic differentiation index F_ST value was less than 0.05, indicating no obvious genetic differentiation among the populations. The re-sults of AMOVA showed that the genetic difference of Bombay duck populations was mainly from the variation among individuals within the population. The values of Tajima’s D and Fu’s F_s tests were both negatively and significantly deviated from neutral test. The nucleotide mismatch distribution showed distinct unimodal, indicating that Bombay duck experienced a population expansion event in history. We estimated that the population expansion occurred about 0.08—0.32 million years ago in the middle and late Quaternary Pleistocene based on the evolutionary rate of Cyt b gene 2%/Ma (millions years) in marine fishes.