Chinese soft-shelled turtle Pelodiscus sinensis has been an important aquaculture species in Southeast Asian countries. To breed a new variety of soft-shelled turtle with excellent properties and to evaluate the effect of hybridization of two turtle strains with a highly different trait phenotype, inheritance, microsatellite loci, and transcriptome analysis were studied in the hybrid turtles and their parents of P. sinensis Japanese strain and Qingxi black turtle. The genotypic characteristics and economic trait of the hybrid turtles were analyzed and compared to the two parents, showing significant growth vigor. The chromosome number of the hybrid turtle was diploid (2N = 66). The karyotype formulae were 8m+10sm+26t+22mc, with little differences between the two parents. Genotypic segregations of 241 microsatellite loci were screened in 3 populations including 90 species and showed that the specific allele numbers and polymorphic fragments increased in hybrid turtles indicating genetic diversity increased by hybridization. The liver transcriptome analysis of the hybrids and two parents showed similar distribution abundance in the parental and hybrid groups, but the transcripts with high abundance appeared in the hybrid group. There were 274 significant differentially expressed transcripts in the hybrid group compared to the two parental groups, among them 7 differentially expressed genes indicating super-parent expression, and only 2 genes showing low-parent expression. In the differentially expressed genes, expression changes were mainly contributed to regulatory region changes rather than coding region sequences. These results would be important for facilitating successful breeding strategies by hybridization in P. sinensis.