The Chinese soft-shelled turtle (Pelodiscus sinensis) is a valuable aquaculture species in China that exhibits typical sexual dimorphism and is classified as GSD (genetic sex determination) species due to the presence of ZZ/ZW micro-sex chromosomes. However, the development and differentiation of germ cells in the gonads of male and female P. sinensis have been rarely studied. To characterize the profile of germ cells in both male and female developmental gonads, we utililzed embryos at developmental stages 14 to 20, which encompassed the sex determination and sex differentiation periods of P. sinensis. Germ cells from stages 14 to 20 were sampled, and their morphology was examined using tissue sections. Immunohistochemistry was employed to detect the expression of related proteins, while RNA was extracted from gonads at stages 14 to 20 for RNA-seq and qPCR analyzes to investigate the differential expression of male and female germ cell-related genes. Germ cell marker genes included Ddx4 and Dazl, while genes associated with reproductive cell growth and development included Mael,Dnd1, Stra8 and Dmc1, Rbm38, Hes1, Notch and Bcl2. The tissue sections revealed that germ cells in the embryonic stage were larger than surrounding somatic cells, with large nuclei and prominent nucleoli in both ZW female and ZZ male gonads. Immunofluorescence staining of DDX4 showed no differences in germ cell distribution between male and female gonads from stages 14 to 17. However, from stage 17 to 20, germ cells in the gonads of ZZ females were concentrated in the cortex, whereas male germ cells were concentrated in the medulla. RNA-seq analysis indicated that Ddx4, Mael, Dazl, Dnd1, Stra8 and Dmc1 were highly expressed in female gonads, while Rbm38, Hes1, Notch and Bcl2 were highly expressed in male gonads during stage 17 to 20. Quantitative Real-time PCR analysis confirmed that mRNA expression levels of the germ cell-related genes Ddx4 and Dazl were significantly higher in ZW female gonads than in ZZ male gonads. Similarly, expression levels of meiosis-related genes Stra8 and Dmc1 were significantly higher in ZW female gonads. These results elucidate the differences in the development of male and female reproductive cells and the timing of differentiation. This study provides an important reference for research on turtle germ cell development and supports sex control breeding efforts in P. sinensis.