The transcription factor sox9 has been implicated in cartilage formation and testis determination in mammals. Here, two duplicates of sox9 were found in Japanese flounder (Paralichthys olivaceus) named Posox9a and Posox9b, respectively. Phylogenetic and gene structure analyses revealed that Posox9a and Posox9b were homologous to that of teleosts and tetrapods. Quantitative real-time polymerase chain reaction (qRT-PCR) showed that both Posox9a and Posox9b expressed higher in testis than in ovary of adult tissues. The in situ hybridization (ISH) analysis of gonads showed that Posox9a and Posox9b mRNA were both detected in oocytes, Sertoli cells and spermatocytes. During sex differentiation, the expression of Posox9a exhibited obvious sexual dimorphic expression from 60 days after hatch (dah) with higher expression in male preferred individuals than female preferred individuals and increased gradually from 30 to 100 dah. A similar pattern was detected in Posox9b expression. After injection of androgen (17α-methyltestosterone) of different concentrations, the expression level of Posox9b increased significantly, whereas Posox9a did not change obviously. These results indicated that the two sox9 genes of Japanese flounder had converse functions in sex differentiation, whereas their differences in 17α-methyltestosterone administration were obvious and worthwhile for exploring evolutionary and adaptive significance. This study provided a foundation for further exploration of the roles of Posox9 genes during the sex determination and differentiation, spermatogenesis and gonadal function maintenance of Japanese flounder.