In order to further explore the Chinese soft-shelled turtle(Pelodiscus sinensis) and provide theoretical basis for the mechanism of sex determination, we improved the preparation of chromosome specimen and used precision instrument to analyse its karyotype. The best chromosome specimens can be gained by the injection of colchicine 1 μg/g (weight), effecting 1 h. The karyotype formula of it is 2 n=66=4 m+4 st+34 sm+22T+ZZ/ZW, NF=104(male) and 105(female). In 33 pairs of chromosomes, the No.1-6 are large chromosomes, and the rest are small chromosomes. The sex chromosome types are ZZ(male) and ZW(female). The G-banding of chromosomes was made by trypsinase method, and the schema chart of it was mapped out.