The present study aims to elucidate the different expression patterns and possible roles of Doublesex and Mab-3-related transcription factor 1 (dmrt1), dmrt4, SRY-related transcription factor 9 (sox9) and cytochrome P450 aromatase 19a (cyp19a) during gonadal differentiation in olive flounder, Paralichthys olivaceus. We first analyzed the gene expression patterns in tissues using RT-PCR, which indicated dmrt1, sox9 and cyp19a were sex-related genes with sexual dimorphic expression. The quantitative expression changes of these three genes together with dmrt4 during gonadal differentiation were further examined using real-time RT-PCR. The results showed that dmrt1 was scarcely expressed in the primitive gonad and during following periods of gonadal differentiation. Its expression increased rapidly in the differentiating testis. Dmrt4 was strongly expressed in primitive gonads and much less expressed during following periods of gonadal differentiation. Its expression became strong in differentiating testes. While sox9 was highly expressed in the primitive gonad, it was expressed with fluctuations during following periods of gonadal differentiation. Cyp19a started expressing in primitive gonads, and its expression quantity fluctuated during latter periods of gonadal differentiation, but was strongly expressed in the early stage of differentiating ovaries. Results of in situ hybridization showed that dmrt4 and sox9 transcripts were both mainly localized in spermatocytes and our results suggested these four sex-related genes might be involved in gonadal differentiation through their synergistic effects in flounder.