In order to analyze the genetic diversity of Protosalanx hyalocranius in the Hongze Lake, the mitochondrial cytochrome b (Cytb) and cytochrome c oxidase subunitⅠ(COⅠ) gene sequences were investigated in this study. Using PCR amplification and sequencing, 1141 bp of Cytb and 630 bp of COⅠ sequences were identified. For the Cytb sequence, the contents of A, T, G and C accounted for 21.7%, 29.3%, 16.7%, and 32.3% respectively. Six polymorphic sites and 7 haplotypes were obtained from 40 individuals. The haplotype diversity and nucleotide diversity were 0.775 and 0.00129 respectively. The average nucleotide difference was 1.469. For the COⅠsequence, the contents of A, T, G and C accounted for 21.6%, 26.0%, 19.2% and 33.2% respectively. Five polymorphic sites and 6 haplotypes were obtained from 40 individuals. The haplotype diversity and nucleotide diversity were 0.700 and 0.00207 respectively. The average nucleotide difference was 1.303. The kimura-two-parameters analysis showed that the genetic distances of the haplotypes varied between 0.001 and 0.004 for Cytb, and between 0.002 and 0.005 for COⅠ. Furthermore, the phylogenetic tree constructed with the neighbor-joining (NJ) method indicated that the haplotypes of P. hyalocranius formed one clade, which suggested that there was no genetic differentiation in the haplotypes of Cytb and COⅠof P. hyalocranius. Fu’s Fs values (–0.32836 for Ctyb and –0.52548 for COⅠ) in the neutrality test was negative, but there was no significant difference. The mismatched bases distribution analysis showed a peak type, which indicated that P. hyalocranius in the Hongze Lake had experienced a population expansion.