Procambarus clarkii is an alien invasive crayfish species which is widely distributed in wetlands in China. Clarification of P.clarkii population genetic structure will help to understand its spread process. Here, we analyzed the population genetic structures of 9 populations of 151 crayfish individuals in Huaihe river based on the COⅠ region. The results showed that the 624 bp long COⅠ region aligned across 25 haplotypes. AMOVA showed that intra-population variation (92.20%) was much higher than inter-population variation (7.80%). Haplotype diversity and nucleotide diversity were moderate, and the differentiation among populations was non-significant (F_st=0.078). Phylogenetic tree shows that the genealogical clusters do not match the sample locations well, suggesting that genetic differentiation among them is not remarkable, while the gene flow is evident.