Abstract: Halfsmooth tongue-sole (Cynoglossus semilaevis Günther) is a cultured marine fish exploited recently in China. The female individuals of tongue sole grow 1-2 times faster than male individuals. Thus, the development of all-female stock would be of significant benefit for aquaculture. Sex-related molecular marker is a useful tool for studying sex determination mechnism and controling fish sex. In order to screen sex specific molecular markers, AFLP analysis technique was firstly developed in half-smooth tongue-sole. DNA extraction from liver tissues was carried out according to stardand procedures. Phenotypic sex of the fish was determined by histological sectioning and staining. 64 AFLP primer-combinations were used to screen the genome DNA of half-smooth tongue-soles. 4 primer-combinations amplified 7 female-specific markers that were female specific in half-smooth tongue-sole. The 7 female-specific markers were named CseF382, CseF575, CseF783, CseF464, CseF136, CseF618 and CseF305, respectively. One female-specific AFLP marker (CseF382) was amplified, recovered from the gels, cloned, and sequenced. This female-specific AFLP marker was converted into single locus PCR marker of a sequence-characterized amplified region (SCAR). A simple PCR method of using the specific primers was developed for identifying genetic sex of half-smooth tongue sole. This PCR method will be more efficient and less expensive than with AFLP markers. The isolation of sex-specific molecular markers lays a basis for elucidation of sex determination mechanism, and provides a tool for sex control in half-smooth tongue sole.