The molecular mechanism of sex determination in Pelodiscus sinensis has been a long-standing mystery.In many vertebrate species,SOX9 gene is required for male sexual differentiation.In this study,the mRNA and protein expression of SOX9 gene and its cellular localization in gonads of different developmental stages of both sexes were examined to investigate the involvement of SOX9 in sexual differentiation in P.sinensis.Real-time PCR results showed that,among different tissues,SOX9 gene highly expressed in testis tissue.Immunofluorescence analysis revealed that SOX9 protein started to express at embryonic stage 18,and localized in the nuclei of pre-sertoli cells in male embryonic gonads.However,no SOX9 protein was detected in female gonads during embryonic development.Moreover,SOX9 protein expression significantly decreased in estrogen-induced male to female sex reversal.Whereas SOX9 protein was triggered to highly express in aromatase inhibitor-induced female to male sex reversal.In conclusion,this study indicates that SOX9 gene is a male-specific gene and participates in the male gonadal development,which is likely to play a key role during primary male sex determination in P.sinensis.