In this study, we compared the extent of genetic diversity of the triploid and diploid flounders using the microsatellite method. The triploid were induced using cold shock method in 2012, and then they were cultured under the same conditions as their diploid control. Genomic DNAs were isolated using the high salt-extraction method from the muscle tissues of 32 triploid and 32 diploid flounders of 11-month old. The genetic variations of the triploid and diploid flounders were assessed based on the selected 21 microsatellite loci. The PCR products were isolated using 14% PAGE electrophoresis, and the microsatellite alleles were artificially identified and analyzed. The results showed that in the triploid and diploid stocks, the average numbers of alleles per locus (A) were 5.6 and 5.0; the total numbers of genotype were 190 and 142; the average numbers of effective alleles per locus (ae) were 3.7 and 2.8; the values of average observed heterozygosity (HO) were 0.613 and 0.868; and the unbiased expected heterozygosity (HE) were 0.681 and 0.629, respectively. The values of the polymorphism information content (PIC), the power discrimination (DP) and the probability of paternity exclusion (PPE) in the triploid and diploid stocks were 0.642 and 0.589, 0.660 and 0.609, 0.482 and 0.399, respectively. The accumulation of PIC and PPE both achieved 0.999, which indicated that these microsatellite loci were very sensitive and could be used in the parentage and kinship determination in future genetic breeding studies. The genetic distance (D), the genetic similarity (I) and the coefficient of gene differentiation (GST) between the triploid and the diploid stocks were 0.312, 0.732 and 0.971 respectively. The genetic diversity of the triploid stock was moderately reduced compared to the diploid stock. There were also some differences in the genotypes between the triploid and diploid flounders.