Hops are dioecious, with only female plants cultivated for resin production and primarily used in brewing. Therefore, early sex identification at the seedling stage is crucial for breeding and cultivation. Molecular marker-assisted selection facilitates rapid and reliable sex identification of hops. We developed molecular male markers by leveraging sequence information from male-specific regions in a designated public database. To assess the accuracy of sex identification using the newly generated markers, we performed PCR analysis on four cultivars of hops with known sexes. Seventy-eight percent of the tested PCR primers correlated with the male sex phenotype. Following optimization, four primer pairs were successfully converted into male-specific PCR markers.