China is currently the largest producer and consumer of red swamp crayfish (Procambarus clarkii) in the world. However, self-propagation breeding has resulted in germplasm degradation, which led to smaller body sizes and weaker disease resistance. Thus, implementing selective breeding to enhance commercially desirable traits is critical issue for the sustainable development of the P. clarkii industry. Maintenance of high genetic diversity within breeding populations is essential for a selective breeding program. Microsatellites are valuable tools for analyzing genetic diversity and structure in aquatic species. In this study, 20 microsatellite markers were analyzed across five consecutive generations (F₁‒F₅) of P. clarkii (n=389). The base population was sourced from wild populations from Jieliang Lake (Hubei Province), Honghu Lake (Hubei Province), and Hongze Lake (Jiangsu Province). The results showed that the number of alleles per loci ranged from 5 to 22, with an average polymorphic information content (PIC) of 0.711, indicating substantial genetic polymorphism suitable for further research. Throughout the five selected generations, the average number of effective alleles (N_e) decreased from 4.216 to 3.815, the average observed heterozygosity (H_o) decreased from 0.539 to 0.433, the average expected heterozygosity (H_e) decreased from 0.734 to 0.680, and the average PIC decreased from 0.695 to 0.638. Although N_e, H_o, H_e, and PIC showed a decreasing trend over successive generations, the differences were not significant (Kruskal‒Wallis test, df=4, P＞0.05). Therefore, genetic diversity was maintained in the selected strains over five generations. The inbreeding coefficient (F_is) ranged from 0.106 to 0.622, with an average of 0.304, and all microsatellite loci were positive, indicating inbreeding within the five successive generations. Genetic distances between F₁‒F₅ ranged from 0.061 to 0.222, with the smallest genetic distance observed between F₁ and F₂ and the largest between F₂ and F₄. The genetic differentiation coefficients (F_st) among the five successive P. clarkii generations were all less than 0.050, suggesting a low level of genetic differentiation. Analysis of molecular variance (AMOVA) revealed that 95.16% of genetic variation occurred within generations, while 4.84% was attributed to differences among generations, suggesting the occurrence of genetic differentiation among the generations. According to the genetic distance data, the cluster tree constructed by unweighted pair-group method with arithmetic means showed that the five generations were divided into two branches. F₁ and F₂ were clustered into one branch, and F₄ and F₅ clustered first before merging with F₃ as another branch. In conclusion, the genetic diversity of each generation was maintained at a relatively high level, indicating that there is still potential for breeding in selected strains of P. clarkii. In future breeding programs, parental populations from diverse genetic backgrounds should be incorporated or the number of breeding individuals should be increased to mitigate inbreeding depression.