The genetic diversity and structure of Protosalanx hyalocranius in four freshwater lakes in Jiangsu Province were assessed. Based on PCR amplification and sequencing technologies, the mitochondrial Cytb gene sequences from 160 individuals of four freshwater lake populations (Taihu Lake, Gaoyou Lake, Hongze Lake and Luoma Lake) were analyzed. The results showed that 25 variable sites were detected in 160 Cytb gene sequences, containing 11 singleton mutation and 14 parsimony informative sites. A total of 23 haplotypes were defined in all 4 populations and overall haplotype diversity (H_d) and nucleotide diversity (π) were 0.878±0.014 and 0.002 1±0.000 1, respectively. Therefore, the genetic diversity of P. hyalocranius showed the pattern of high haplotype diversity and low nucleotide diversity. In the four populations, the Gaoyou Lake and Taihu Lake populations had the highest haplotype diversity (0.871±0.031) and nucleotide diversity (0.002 9±0.000 1), respectively, and the Hongze Lake population had the lowest haplotype diversity (0.755±0.455) and nucleotide diversity (0.001 3±0.000 1). The result of AMOVA showed that 15.13% molecular variation was among populations and 84.87% within populations, and most molecular variation occurred within populations. The pairwise fixation index (F_st) revealed that there were significant differences between Taihu Lake population and Gaohu Lake, Hongze Lake and Luoma Lake populations, but there were no significant differences among Gaohu Lake, Hongze Lake and Luoma Lake populations. It is concluded that the Taihu Lake population and the other three lake populations should be managed and protected as different evolutionary units. Both the mismatch distribution analysis and the neutrality tests indicated that there was a recent population expansion in P. hyalocranius, which might have occurred 103 000-41 000 years ago during the late Pleistocene. Despite the high genetic diversity, P. hyalocranius already showed obvious signs of decline. More attentions should be paid to the management of fisheries in order to protect the germplasm resources of P. hyalocranius and to maintain the sustainable development.