Abstract: The Asian giant soft-shelled turtles (Pelochelys cantorii) belong to Testudines, Trionychidae, and Pelochelys. As one of the largest aquatic turtles in China, it is also an important indicator species for the health of the river ecosystem in the Pearl River Basin and even the southern part of China. Because of the long history of P. cantorii, it is of great scientific value of environmental changes and biological evolution. P. cantorii was widely distributed in Southeast Asia of China in history. However, due to unfair trade, excessive harvesting and killing of turtles and water conservancy projects, the population of P. cantorii has been greatly reduced. Currently, P. cantorii have become an extremely endangered species. There are only 13 P. cantorii in captivity in China, which have been listed as national first-class protected animals and taken seriously by scientific researchers. With the success of artificial domestication and breeding in recent 3 years, the genetic information of the preserved population is still unknown. In this paper, we use transcriptome data design trinucleotide repeat and repeat four nucleotides microsatellite primers 30000 on, the choice of 230 pairs of primer synthesis, received 10 after two screening amplification with high efficiency and high specificity of microsatellite marker, and build 2 multiple PCR system. Genotypes and genetic diversity of 56 soft-shelled islet were detected by ABI3130 genetic analyzer and PopGene3.2 and CERVUS Software. The results showed that the average number of alleles was 2.3 and the average content of polymorphic information was 0.3829. The average observed and expected heterozygosity were 0.6305 and 0.4767, respectively. Paternity analysis of 56 progenies showed that the cumulative exclusion probabilities of 10 microsatellite loci were 73.14% (NE-1P), 90.98% (NE-2P) and 98% (NE-3p), which can meet the needs of paternity testing, respectively, and the four parents showed differences in reproductive selection. The analysis of the technology can help identify the captive population of genetic information, assist researchers to family management of groups. And more importantly, in the process of F₃ generations of breeding in the future, the technology can be regarded as parental choice of technical means, to get healthier, higher genetic diversity, and their offspring, for the entire group of proliferation and endangered wild work to lay a solid foundation.