Culter alburnus has high nutritional, economic and ecological values, but its wild resources tend to be exhausted in recent years. So, it is urgent to protect germplasm resources of C.alburnus. In this study, the molecular marker of mtDNA Cyt b gene was used to investigate the genetic diversity and genetic structure of C.alburnus populations in Tai Lake and Hongze Lake, and the Cyt b gene sequences were amplified and sequenced. The research results showed that the full length of Cyt b gene was 1 141 bp, and the base content of A+T was higher than that of G+C.29 variable sites were detected and 29 haplotypes were defined in 98 Cyt b sequences. The average haplotype diversity (Hd) and nucleotide diversity (Pi) of C.alburnus were 0.923 and 0.002 74, respectively, and the average number of nucleotide differences was 3.127. The Hd values of Tai Lake and Hongze Lake populations were 0.927 and 0.816, respectively, and the Pi values of two lake populations were 0.002 85 and 0.002 20, respectively. The genetic diversity of C.alburnus showed high Hd and low Pi. The results of molecular variance analysis showed that the proportion of molecular variation within populations was 15.01%, and the proportion of molecular variation between populations was 84.99%. The coefficient of genetic differentiation (Fst) was 0.150 15 (P<0.01), indicating that there was significant genetic differentiation between the two populations. The values of Tajima's D and Fu's Fs neutral tests were both negative, but significant difference was only found for Fu's Fs neutral test. Furthermore, the mismatch distribution curve was obvious unimodal. Both neutral test and mismatch distribution suggested C.alburnus had experienced population expansion in evolutionary history. In a word, the present results could provide scientific data for the management and protection of C.alburnus germplasm resources in Tai Lake and Hongze Lake.