Abstract: To isolate and identify polymorphic microsatellite markers which would be used for evaluating the genetic diversity of Chelydra serpentina L. An enriched microsatellite library of C. serpentina was constructed by the magnetic-bead enrichment method used biotinylated (CA)15 probe. Screened the enriched microsatellite library by PCR amplification technique, seventy microsatellite sequences were obtained. To analyze genetic diversity of a cultured C. serpentina, forty eight pairs of primers were designed on the basis of the microsatellite sequences and 36 of that were screened by PCR amplification technique. In these microsatellite loci, the number of alleles per locus ranged from two to nine with an average of 4.361. The number of effective allele ranged from 1.461 to 7.767 with an average of 3.498. The size of allele fragment ranged between 56 and 342 bp. The observed heterozygosity varied from 0.067 to 1.000 with an average of 0.725. The expected heterozygosity varied from 0.316 to 0.850 with an average of 0.600. Polymorphism information content (PIC) ranged from 0.2655 to 0.8359 with an average of 0.5573. The average fixation index coefficient of population was -0.214, which suggested surplus heterozygotes in the population. These results indicated that the cultured C. serpentina populations have relatively high genetic diversity.