Genetic structure and phylogeography of seventeen natural populations of Macrobrachium nipponense in Huaihe River were analyzed based on mitochondrial Cytochrome Oxidase Subunit I (COI) gene sequence. Total DNA was extracted from 10-19 individuals of each population. A fragment of the 623 bp COI gene was amplified using the primers LCO1492 and HCO2198. 48 variable nucleotides were detected, and 31 haplotypes with 12 shared haplotypes were found in 17 populations. The result of haplotypic and nucleotide diversity analysis showed a middle genetic diversity. AMOVA analysis across all populations showed that there were low levels of genetic divergences among the 17 populations (F_st = 0.041 3, P<0.05). The Kimura 2-paramter genetic distance between Wuhe and Jiaogang, Huajia, Wabu populations (D_A= 0.014) was higher than that between other populations. UPGMA tree and haplotype network based on the 31 haplotypes indicated that the populations of the lower Huaihe River formed one clade and the populations of the middle Huaihe River formed the two others. The biomodel mismatch distribution of pairwise haplotype difference and the neutrality tests showed that population expansion occurred. These results would offer theoretical direction for the protection and utilization of the M.nipponense in Huaihe River.