Macrobrachium nipponense is a freshwater prawn with high economic value. It is widely distributed in most freshwater water bodies and in all river systems of Henan Province. With the development of aquaculture, the change of ecological environment in the water area, and the influence of contrived factors, the germplasm resources of M. nipponense have also been gradually affected in recent years. To provide a reference for M. nipponense in Henan Province, we assess the germplasm resources and genetic diversity of M. nipponense in Henan Province. The mitochondrial COI gene was used as a marker to investigate population genetics of 135 M. nipponense samples from 18 populations in Henan Province. The samples covered four main water systems in Henan Province, including the Yangtze, Yellow, Huaihe, and Haihe Rivers. Results showed that the 18 populations of M. nipponense in Henan Province contained 27 haplotypes. The average haplotype diversity (H_d) and average nucleotide diversity (π) were 0.78574 and 0.01059, respectively. The Huaihe population had the lowest genetic diversity, and the Yangtze River population showed the highest genetic diversity. High Hd and low π were observed for the four rivers. AMOVA showed that major genetic variation (66.03%) occurred within the population, and 40.22% of the genetic variation existed among populations. There was no significant genetic differentiation for M. nipponense from the four water systems based on phylogenetic and haplotype network analyses. The demographic history analysis showed that M. nipponense of Henan province did not experience recent population expansion, maintaining a relatively stable population size. This study can provide basic guidance for protecting and reasonably utilizing the germplasm resource of M. nipponense in Henan Province, providing reference material for the whole of China.