We isolated a Sox9 homologue, termed Sox9b, from adult allotetraploids. On the basis of the HMG-box amino acid sequences, Sox9b can be categorized into the same subgroup of Sox-E proteins as SOX8, 9, and 10. Interestingly, Northern and Southern hybridization demonstrated that Sox9b was highly expressed in the testis but at lower levels in the heart and brain, while no expression of Sox9b mRNA was discovered in the ovary of adult allotetraploids. Two transcripts of Sox9 (2.1 kb and 1.7 kb) with overlapping expression were observed for the first time in teleosts. These results indicated that allotetraploid Sox9b might be involved in gonadal development of male allotetraploids. In addition, the diverse tissue-specific expression profiles of Sox9b provided significant molecular evidence for the lineage-specific subfunction partitioning of Sox9 genes.