Verasper variegatus, a marine flatfish, holds significant nutritional and economic value and exhibits sexual dimorphism in growth, with females growing faster than the males. However, to date, no rapid, effective, and universal sex-specific DNA marker has been available for genetic sex identification and sex-controlled breeding in V. variegatus. To address this, we performed whole genome resequencing on 17 male and 17 female individuals to identify female-specific DNA sequences. Through PCR amplification, we identified a female-specific DNA marker and established a genetic sex identification method. Our results revealed 359 potential female-specific DNA sequences following reads mapping, sequencing depth, and coverage analysis. Primer pairs targeting approximately 150 bp DNA sequences flanking these regions were designed. After two rounds of PCR verification, a 69 bp insertion was identified as a female-specific marker, accurately distinguishing genetic sex via PCR amplification and 2% agarose gel electrophoresis. Further validated using 117 individuals from another breeding population confirmed the accuracy of this marker and method, with genetic sex results aligning with phenotypic determined by gonad histology. The development of this female-specific DNA marker and the genetic sex identification method provides crucial genetic insights into the sex determination mechanism of V. variegatus and accelerates the implementation of sex-controlled breeding and all-female breeding techniques.