Abstract: In order to elucidate the mechanisms of sex determination and differentiation in Culter alburnus and to control breeding, this study analyzed the role of C. alburnus Sox9 during gonad development. The cDNA sequence of two homologous genes Sox9a and Sox9b were obtained. The full-length cDNA of Sox9a was 1642 bp, encoding 458 amino acids; the full-length cDNA of Sox9b was 1673 bp, encoding 456 amino acids. Sequence analysis revealed that the similarity between them was 73.95% and the HMG motif regions were highly conservative. Protein second structural prediction showed that Sox9a and Sox9b contained a HMG domain and two nuclear localization signal (NLS) sequences; their three-dimensional structure contained multiple spiral structures. Phylogenetic tree analysis discovered that C. alburnus Sox9a was the closest to Oreochromis niloticus, while Sox9b formed a separate branch. Sox9a was highly expressed in brain and testes, followed by muscle, fins, eyes and ovaries, and rarely detected in kidney, spleen and liver. The expression of Sox9b were only detected in brain, fins, eyes and testes. Promoter CpG methylation analysis of Sox9a showed that CpGs were not methylated in the testes, whereas CpGs were hypermethylated in the ovaries. These results suggested that promoter CpG methylation can regulate sexual dimorphic expression of sex-related genes, and epigenetic modification may play an important role in the gonad development of C. alburnus.