Ayu (Plecoglossus altivelis) is a valuable economic fish in Aisa. The difference between males and females is obvious. Females have better growth traits than males, with faster growth and larger individuals. The market price of female fish is more than twice that of male fish. Therefore, the identification of the sex of ayu is extremely important to the economic value of breeding. To identify potential Sex-specific markers in male and female ayu genome, we used a modified 2b-RAD genotyping method to find putative SNP loci and Inserts/Deletions (InDels) that may be associated with each sex. In this study, we constructed 2b-RAD libraries for ten sires and ten dams of ayu with known sex phenotypes (brood fish).