The Chinese mitten crab, , is one of the most important aquaculture species in China, and is widely distributed in Eastern Asia. This study was conducted to evaluate the genetic diversity and genetic structure of cultured and wild populations of River, and Liaohe River based on the mitochondrial DNA D-loop region. The results showed that the length of the D-loop gene segment used in this study was 477 bp, which contained a total of 234 variation sites and 131 parsimony information sites for all tested samples. The 262 samples from six populations had 110 haplotypes, which consisted of 90 unique haplotypes and 20 shared haplotypes. The haplotype diversity index () of the six populations ranged from 0.88889 to 0.96522, and the nucleotide diversity index () ranged from 0.00887 to 0.01602. The genetic diversity levels of cultured populations were HC > YC > LC, whereas the levels of wild populations were HW > LW > YW(LC and LW indicate cultured and wild population of Liaohe River respectively, HC and HW indicate cultured and wild population of Yellow River respectively, YC and YW indicate cultured and wild population of Yangtze River respectively). The genetic distance () ranged from 0.0119 to 0.0173 for the six populations. Regardless of their wild or cultured status, the genetic distance between the Liaohe and Yangtze populations was the smallest among three rivers. The genetic differentiation index () was 0.12938 among the six populations. A neutral test of the six populations showed the values of Tajima's were negative. The population's Tajima's value test showed that these groups have a complex population history, and it is speculated that has had a large-scale group expansion event. The genetic distances of the six populations were analyzed. It was found that the overall genetic distance was not large. The results of genetic differentiation showed that there was no significant difference between the LW and YC, and YC and YW populations. The results of the AMOVA analysis of the six populations showed that population genetic differences mainly occurred in the six population. The phylogenetic trees may have resulted from of a certain degree of confounding caused by the cross-regional introduction of the Chinese mitten crab population in the Liaohe River system. These results provide valuable information for the evaluation, protection, and exploitation of resources.