The invention discloses a microsatellite DNA (Deoxyribonucleic Acid) markers of a Chinese three-keeled pond turtle, which are determined by comprising the steps of constructing a library rich in tortoise microsatellites (CA)n (Carbonic Anhydrase)n and (GATA)n, screening and sequencing positive clones containing microsatellite sequences to obtain 59 clones containing microsatellite repetitive sequences, and determining 10 microsatellite markers with rich polymorphisms, wherein the 10 microstaellite markers are Mclw01, Mclw02, Mclw03, Mclw04, Mclw05, Mclw06, Mclw07, Mclw08, Mclw09 and Mclw10. The invention provides 10 new microsatellite loci of the Chinese three-keeled pond turtle and primer sequences and amplification methods for amplifying the 10 microsatellite loci, can be applied to a study on genetic diversity of different geographical populations of the Chinese three-keeled pond turtles and a study on paternity identification, and the molecular markers are reliable and effective with good repeatability.