Molecular sex identification is important in studying sex control, sex determination, and all-female breeding in half-smooth tongue sole (Cynoglossus semilaevis). In the present study, a female-specific AFLP marker was isolated from Cynoglossus semilaevis by AFLP technique using the selective primer combination E-ACT/M-CAA. This marker was re-amplified, recovered from the agarose gels, cloned and sequenced. Bioinformatic analysis indicated that the length of the product was 791 bp, and the sequence showed no similarity to any known sequences deposited in the GenBank database using BLASTn. According to the DNA sequence of the female-specific AFLP marker, specific PCR primers were designed and PCR amplification was performed on 100 sex-known individuals of C. semilaevis (50 females and 50 males each). A specific band 324 bp in length was present in all females but absent in all males (except for one male), indicating that the female-specific AFLP marker was successfully converted into female-specific SCAR (sequence characterized amplified regions) marker. The sex analysis of 3-day-old C. semilaevis individuals using this female-specific SCAR marker indicated that the female ratio was 41.7%. The female-specific SCAR marker developed in this study allowed simple, reliable, and rapid molecular sex identification using small amounts of fin tissue without sacrifice of C. semilaevis especially at early stage of development.