Pacific threadfin, Polydactylus sexfilis, a highly desirable food fish in Hawaii, has been raised by offshore-cage culture in a large scale. To develop an effective genetic tool to monitor the broodstock resources and their mating patterns, we identified microsatellite loci of Pacific threadfin. Genotyping and analysis of 10 microsatellite loci in the wild and captive broodstock populations did not indicate significant differences among these populations. Five polymorphic microsatellite loci were further selected for the development of a parentage assignment test. Parental assignment analysis of one broodstock population showed that 90% of the offspring from three consecutive spawning events were successfully assigned to their specific parent-pairs. These five microsatellite loci provide an accurate and cost-effective method to assess the genetic variability and mating patterns in Pacific threadfin.