Portunus trituberculatus is one of the most commercially important marine crustacean species for both aquaculture and fisheries in Southeast and East Asia. Production of monosex female stocks is attractive in commercial production since females are more profitable than their male counterparts. Identification and mapping of the sex-linked locus is an efficient way to elucidate the mechanisms of sex determination in the species and support the development of protocols for monosex female production. In this study, a sex-averaged map and two sex-specific genetic maps were constructed based on 2b-restriction site-associated DNA sequencing. A total of 6349 genetic markers were assigned to 53 linkage groups. Little difference was observed in the pattern of sex-specific recombination between females and males. Association analysis and linkage mapping identified 7 markers strongly associated with sex, four of which were successfully mapped on the extremity of linkage group 22. Females were homozygous and males were heterozygous for those 7 markers strongly suggesting an XX/XY sex determination system in this species. Three Markers were successfully validated in a wild population of P. trituberculatus and exhibited a specificity ranging from 93.3% to 100%. A high-resolution melting based assay was developed for sex genotyping. This study provides new knowledge and tools for sex identification which will help the development of protocols for monosex female production of P. trituberculatus and support future genomic studies.