To elucidate the role of sox9 in sex determination and differentiation of the large yellow croaker (Larimichthys crocea), the full length of sox9a and sox9b were cloned using reverse transcription-polymerase chain reaction (qRT-PCR) and rapid amplification of cDNA ends (RACE). The difference of the gene expression in various tissues and development stages was analyzed through quantitative real-time PCR. Expression profiles of sox9a/b after 17β-estradiol or 17α-methyl testosterone treatments were also examined. The full-length cDNA of sox9a gene is 2 442 bp (NCBI:MH996431), including a 476 bp 5' UTR, a 466 bp 3' UTR and a 1 500 bp ORF coding a polypeptide of 499 amino acids. The full-length cDNA of sox9b gene is 2 199 bp (NCBI:MH996432), including a 335 bp 5' UTR, a 415 bp 3' UTR and a 1 449 bp ORF coding a polypeptide of 482 amino acids.Quantitative Real-time PCR results showed that sox9a was primarily expressed in gonad, eye, brain, liver, and the expression level in testis was significantly higher than that in ovaries. sox9b was widely expressed in multiple tissues in large yellow croaker; the expression level was the highest in testes, but can be barely detected in ovaries. At early developmental stages of fry, sox9a/b was expressed at a lower level. Sox9a/b peaked at 84 dph (day post hatch) and 123 dph, then their expression declined and gradually rose again at 10 mph. In addition, 17β-estradiol can significantly down-regulate the expression of sox9a and sox9b in testes. 17α-methyl testosterone can significantly elevate the expression of sox9a and sox9b in gonads. The study demonstrated that sox9a/b may play important roles in sex determination and differentiation in the large yellow croaker. However, the functions of the two genes may be different.