Carassius auratus var. dongting originates from Beimin Lake, Hunan. Compared to other Carassius spp., C. auratus var. dongting had a higher growth rate and larger body size, and we therefore hypothesized that these growth advantages were related to polyploidization. To verify this hypothesis, a 9 microsatellite ploidy identification method was developed to examine the ploidy level of C. auratus var. dongting, and flow cytometry assays and chromosome karyotype assays were used to further confirm the findings. The results showed that 9 microsatellites could completely distinguish 37 ploidy-known specimens. The genotyping results showed that all the 136 ploidy-unknown individuals were triploid, all three genotypes (AAA, AAB/ABB, and ABC) were observed in this population. The mean DNA content of blood cells in C. auratus var. dongting (159.42±5.64) was 1.5 times that of blood cells in C. auratus var. red (102.43±3.54), and the results from microsatellite markers were totally concordant with those from flow cytometry analysis. The karyotype analysis revealed that the number of chromosomes was 3n=156±and the karyotypic formula was 3n=39m+36sm+81sta, NF=231. This study not only established a microsatellite-based system for ploidy identification of C. auratus, but also systematically confirmed that C. auratus var. dongting is triploid.