Spotted knifejaw (Oplegnathus punctatus), an economically important species in marine aquaculture, employs a unique sex determination mechanism based on a complex sex chromosome system (XXXX/XXY). Males (2n = 47) possess one fewer chromosome than females (2n = 48), and their karyotype includes an unusually large neo-Y chromosome. Additionally, a pronounced sexual dimorphism in growth rate is observed, with males exhibiting a faster growth rate than females. In this study, we conducted a comprehensive whole-genome scan, which initially revealed structural variations in the anti-inflammatory itih4 gene between male and female O. punctatus. Additionally, we designed a pair of primers to detect DNA sequence variations within the itih4a/itih4b gene. These variations are located in the intergenic region of the fusion Y chromosome in male O. punctatus, compared to the homologous X chromosome in females. In females without DNA insertions in the itih4a/itih4b intergenic region, a single band of 351 bp is amplified. By contrast, in males with DNA insertions, two bands are amplified (755 bp and 351 bp). The 755 bp band specifically indicates the presence of a DNA insertion in the itih4a/itih4b intergenic region on the Y chromosome, associated with male-specific genetic traits. Our study will facilitate the rapid identification of the genetic sex of both male and female O. punctatus individuals.