Macrobrachium nipponense, is an important economic indigenous prawn in China, which is widely distributed in freshwater in China, Japan and Southeast Asia. In this study, D-loop region sequences was used to investigate the genetic diversity and population structure of 22 wild populations of M. nipponense through China, containing the major rivers and lakes of China. Totally 475 valid D-loop sequences from 22 populations with a length of 1110bp were obtained, and 348 variation sites and 221 haplotypes were detected. A total of 221 haplotypes were defined in 22 populations. Hap48 was the dominant haplotype, accounting for about 13%. The haplotype diversity (h) was ranged from 0.1630 (BN) ~1.0000 (AR) and the nucleotide diversity π value ranged from 0.001164 (MR) ~ 0.037168 (NR). The pairwise genetic differentiation index (FST) ranged from 0.00344 to 0.91243 and most pair-wised FST was significant (P < 0.05). The lowest FST was displayed in MR and JLR population and the highest was between NDR and NR. The phylogenetic tree of genetic distance based on UPGMA showed that all populations were divided into two branches. The DC, NDR, JLR and MR populations were clustered into one branch. The neutral test and mismatch distribution results showed that M. nipponense populations were not experienced expanding and kept a steady increase. Taken together, a joint resources protection and management strategy for M. nipponense have been suggested based on the results of this study for its sustainable use.