Simple SummaryHigh-quality seedlings are a prerequisite for healthy aquatic animal breeding. As the scale ofProcambarus clarkiifarming continues to expand, the model of “breeding and catching large and keeping small” has led to serious degradation of the fry. This has restricted the development of theP. clarkiiindustry. Therefore, the selection and breeding of optimal breeds ofP. clarkiiare very important. Investigating intraspecific patterns of genetic diversity may help unravel the species’ genetic structure, evaluate the quality of the fry, and design measures for breeding and conservation. In this study, the genetic diversity ofP. clarkiifrom three regions of China was analysed using microsatellite markers. Populations ofP. clarkiishowed high genetic diversity and extensive gene flow. The Xuancheng population could be used as a selective breeding population because it possesses the highest diversity. The results of this study provide a scientific basis for the conservation of germplasm resources and the genetic selection and breeding ofP. clarkii.AbstractWith the increasing scale of crayfish breeding, the self-propagation and “catch large and keep small” breeding patterns have led to serious degradation of the fry, so the selection and breeding of high-quality fry is very important. Selecting a population with a high genetic diversity as the base population for breeding can greatly improve the breeding efficiency. Fifteen microsatellite loci were used to understand the genetic structure and diversity of threeProcambarus clarkiipopulations in Chongming, Shanghai; Gaoyou, Jiangsu; and Xuancheng, Anhui. The results indicated that the three populations were diverse and the number of alleles, observed heterozygosity, expected heterozygosity, Shannon information index, and polymorphic information content ranged from 4.8 to 6.2, 0.5567 to 0.6257, 0.6166 to 0.7086, 1.1292 to 1.3987, and 0.5446 to 0.6452, respectively. The Xuancheng population had the highest genetic diversity. The genetic differentiation coefficient and gene flow of the three populations were between 0.0553 and 0.1068 and 2.0908 and 4.2708, respectively, and there was extensive genetic exchange between the Chongming and Xuancheng populations. Analyses of molecular variance indicated that the genetic variation was mainly within the population (91.51%) and inter-population genetic variation accounted for 8.49%. The unweighted pair group method with an arithmetic mean clustering map was utilised based on the genetic distance between groups, and the results showed that the Gaoyou group was grouped alone, while the Chongming and Xuancheng groups were clustered together. The structural results indicated that the Chongming and Xuancheng groups had the same origin, although the Xuancheng group possessed a more complex genetic structure. This study indicated that all three populations had a high genetic diversity, with the Xuancheng population exhibiting the highest diversity. The results of the study provide a reference for the selection of base populations in breeding programs and confirm that the Xuancheng population in Anhui has a better genetic background. The selection of the Xuancheng population as one of the base populations for genetic breeding will be more efficient to accumulate superior traits.