Abstract: This study investigated the genetic diversity and population demographic history of Hemiculterella sauvagei based on the sequence analysis of mtDNA (Cyt b) of 168 individuals from five geographical populations including Shuiliao, Maotai, Erhe, Taiping and Chishui. After alignment, 1137 bp sequence including 42 variable sites, 13 singleton sites, 29 parsimony informative sites and 38 haplotypes were acquired and revealed, among which Hap1, Hap2 and Hap6 were shared by all populations. The haplotype diversity (H_d) and nucleotide diversity (P_i) were 0.895±0.012 and 0.00487±0.00695, respectively. Analysis of molecular variance (AMOVA) indicated that genetic variations between different geographical populations in the Chishui River were mainly within group. Genetic differentiation index (F_st) indicated high genetic differentiation between Chishui and Maotai, Erhe and Shuiliao population as well as medium genetic differentiation between Chishui and Taiping population. The overall mean genetic distances among five populations were between 0.004—0.006. Tajima’s D and Fu’s F_s neutral tests showed that historical population expansion observed in Taiping, Shuiliao and Chishui population but not in Maotai and Erhe. The population expansion of Shuiliao, Taiping and Chishui appeared in 0.43 Mya, 0.40 Mya and 0.37 Mya, respectively.