Significant differences exist in the growth rate and body size of male and female spotted halibut (Verasper variegatus), with the females being significantly larger than the males. All-female cultivation can improve breeding efficiency. Studies investigating the mechanism of sex determination and features of sex differentiation may provide valuable information for all-female cultivation. In this study, we successfully identified the sox9 gene of spotted halibut. The total length is 3287 bp, including a 1431 bp ORF, encoding 477 amino acids; the 5¢ UTR is 368 bp and the 3¢ UTR is 1488 bp. Polyadenylic acid tails and AATAAA caudal signals were identified in the 3¢ UTR. The expression of the sox9 gene differed in the brain, eye, gills, heart, liver, gallbladder, intestine, testis, ovary, kidney, and muscle in spotted halibut. Transcript levels were high in the gill, brain, and testis, and were significantly higher in the testis than in the other tissues. A two-phase gender difference was observed in the gonads; sox9 expression was significantly higher in the testis than in the ovary, indicating that sox9 might be associated with male gonad development. The dynamic expression patterns of the sox9 gene in spotted halibut larvae gradually declined from 20 to 50 d after hatching, with a sudden rise observed after 60 d. This increased expression might be associated with differentiation of larval gonad.