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.