Abstract: Largemouth bass (Micropterus salmoides) has been introduced approximately for forty years, and it becomes an economically important cultured fish in China. However, there is a lack of natural population supply in breeding and a risk of genetic degradation in the utilization progress. The imbalance between large industrial demand and shortage of improved varieties of M. salmoides are arisen in China. Genetic evolution is the baseline for genetic improvement and selection breeding programs. The microsatellites with multiplex PCR method is popularity applied for genetic analyses in fish species. In this study, we developed six multiplex PCR panels based on 18 polymorphic microsatellites, and subsequently used in genetic analyses for three populations of M. salmoides, i.e., original population of American (USA), “Youlu No. 1” variety (YLO), and their hybrid population (HYB). The results showed that, the parameters of number of alleles (N_a), effective number of alleles (N_e), observed heterozygosity (H_o), expected heterozygosity (H_e) and polymorphism information content (PIC) for 18 loci were ranged from 3 to 14, 1.622 to 5.841, 0.333 to 0.806, 0.361 to 0.810, mean with 7.722, 3.056, 0.577 and 0.579, respectively. Excluding two loci deviated from Hardy-Weinberg equilibrium, the lowest values of all diversity parameters were observed in YLO, while the highest H_o was detected in HYB (0.743), and the highest values of other diversity parameters were detected in USA. The farthest Nei’s genetic distance was evaluated between USA and YLO (0.362); meanwhile, similar Nei’s genetic distances were evaluated between HYB with other two populations (i.e., 0.112 and 0.179 for USA and YLO, respectively). Extremely significant genetic differentiations were revealed among all pairwise populations (P<0.01), and the highest fixation index was detected between USA and YLO (F_ST=0.209). According to the genetic structure analysis based on the allele frequency, relatively independent genetic structures were presented within USA and YLO, while admixture in genetic structure was generally showed in HYB. Additionally, the distinct distributions of individuals from three populations were visualized by using scatter plots of discriminant analysis of principal component (DAPC), and HYB showed with interval distribution between USA and YLO. In brief, six multiplex PCR panels of 18 microsatellites were developed firstly in M. salmoides, relatively high polymorphisms were detected among these loci, and the effective application in genetic analyses was evaluated in this study. That provided valuable tools for genetic and breeding researches in M. salmoides.