The discovery of genetic markers linked to physiological traits in wild populations is increasingly desired for ecological and evolutionary studies, as well as to inform management decisions. However, identifying such markers often requires a large investment of both time and money. Serendipitously, in a recent microsatellite survey, we discovered three out of 16 microsatellite loci that were correlated to the female sex in Pacific halibut (Hippoglossus stenolepis). These three loci were screened in 550 Pacific halibut to determine their accuracy at identifying sex. Genetic assignment successfully identified sex in 92% of individuals from sample collections spanning 3,000 km and 9 years. All but two of 287 females had one copy of a characteristic allele for at least one of the three microsatellite loci, resulting in consistent heterozygote excess in females. This pattern is consistent with the hypothesis that females are the heterogametic sex in Pacific halibut, which thus may have a different sex-determination pattern than the closely related Atlantic halibut (Hippoglossus hippoglossus). A rapid divergence of sex-determining mechanisms could be either a cause or consequence of speciation between Pacific and Atlantic halibut. In either case, the ability to genetically identify sex in individual Pacific halibut provides a new tool for ecological studies, fisheries management, and insight into the evolution of sex determination in flatfish.