The mitochondrial Cytb genes were sequenced and analyzed of W. pigra Whitman from three wild geographical populations including Gaoyou Yangzhou, Zhangjiagang Suzhou and Hailing Taizhou. The results showed that Cytb sequence of W. pigra was 1 146 bp, and the average contents of A, G, T and C were 27.57%, 15.77%, 43.49% and 13.17%, respectively. The A+T content (71.06%) was higher than that of G+C(28.94%).Among examined 77 individuals, 96 polymorphic sites and 58 parsimony information sites were detected. Besides,47 haplotypes were found and there was only one shared haplotype. The total haplotype diversity, nucleotide diversity, and the average nucleotide difference were 0.978 1, 0.013 3, and 15.20, respectively. The Hailing Taizhou population has the highest haplotype diversity (0.987 7) and nucleotide diversity (0.013 1). The genetic differentiation index F_st of the three populations ranged from 0.065 1 to 0.175 6, and the difference was significant (P<0.05), which indicated that there was a certain genetic differentiation among different populations. The analysis result of molecular variance (AMOVA) showed a higher percentage of 88.87% within-population variation than that of 11.13% among-population variation. The haplotype neighbor-joining (NJ) tree with individuals of different geographical groups radiated showed no phylogeographic structure among three populations. Neutral test and mismatch distribution analysis revealed that the overall size of the three populations of W. pigra remained relatively stable, with no significant population expansion.