Several evolutionarily conserved classes of transcriptional regulators were involved in diverse sex determination and differentiation pathways across taxa, whereas their roles in most mollusks is still limited. The Pacific oyster Crassostrea gigas, a dioecious bivalve with sex reversal, could be an ideal model for this issue because of its complex sexuality and potential disruption of sex differentiation in triploid individuals. Here, two mRNA splicing isoforms of a DM domain gene CgDsx and two isoforms of a novel sex-related CgBHMG1 (ortholog of BHMG1 in mammals) were identified in C. gigas. Real time PCR showed that two isoforms of CgDsx and one isoform of CgBHMG1 displayed male-specific expression in diploid oysters, opposite with the female-specific CgFoxl2 (a potential factor of female gonadic differentiation). Interestingly, the four sex-specific transcripts in diploid oyster were expressed in triploid oysters with opposite sex, triploid hermaphrodites and individuals at stage I that sex could not be determined. Subsequent in situ hybridization analysis on gonads of diploid oysters revealed predominant expression of CgDsx in spermatogonia of testes, CgBHMG1 in spermatocytes of testes and follicle cells of ovaries, and CgFoxl2 in follicle cells of ovaries and some male germ cells in testes. And aberrant co-expression of the three genes in triploid oysters was localized in gonadal tubules of gonads at stage I, ovarian follicle cells and undetermined gonial cells in nontypical hermaphroditic gonads with rare female materials. From the above, temporal and spatial expression of sex-related genes in diploid and triploid gonads indicated that CgDsx and CgFoxl2 might mainly function in C. gigas sex differentiation, and CgBHMG1 appeared as a factor involved in meiosis. This work will help to illuminate the gene network of sex differentiation in bivalves and provides new sight on this issue from comparison between diploid and triploid individuals.