Rock bream ( Oplegnathus fasciatus ) is a valuable commercial marine teleost species, which exhibits sexual dimorphism in growth performance. However, the absence of a rapid and cost-effective sex identification method based on sex-specific genetic marker has impeded study on sex determination mechanisms and breeding applications. In the present study, we firstly developed the PCR method for identifying potential sex-specific sequences in Oplegnathus fasciatus with the next-generation sequencing. Sex-specific genomic regions/loci for sex determination were discovered on Chr2 and Chr6 by genome-wide association analysis, sequencing depth, and heterozygosity comparison between females and males. Candidate sex-determining genes ( CCDC63 , ITR , WNT4 ) were furtherly detected in transcriptome data of testes and ovaries. Taken together, a male-specific 34-bp deletion on the Chr2 was identified and developed into molecular marker of sex for O . fasciatus . After validation in individuals with known phenotypic sexes, the accuracy was 100%. This study gives an insight into the mechanism of sex determination in O . fasciatus , and the gender marker is crucial both for future genomic research and for development of efficient and sustainable aquaculture practice.