Identifying small, isolated populations is a conservation priority, not only because isolation may result in negative fitness consequences, but these populations may also harbor unique genetic diversity. The Greater Sage-Grouse (Centrocercus urophasianus) is a widespread obligate species of the sagebrush biome in western North America that has experienced range-wide contraction over the past century. To prevent local extirpation, efforts have been made to identify isolated populations. Here, we analyzed 16 microsatellite loci from 300 Greater Sage-Grouse individuals to assess genetic structure among populations in Wyoming and southeast Montana, particularly with the Jackson Hole and Gros Ventre populations in northwest Wyoming. Four genetic clusters were observed with Pinedale (central-west) and Casper (central) populations forming a cluster, Powder River Basin (central-north) and southeast Montana forming a second cluster, and both Jackson Hole and Gros Ventre forming distinct population clusters. All but the Jackson Hole and Gros Ventre genetic differentiation correspond with designated ecoregions and possessed an isolation-by-distance pattern of differentiation. Both Jackson Hole and Gros Ventre were identified as separate populations with asymmetrical dispersal into Gros Ventre. Both populations also possessed significantly reduced genetic diversity and low effective number of breeders (N_b). Because both populations are surrounded by extensive forested mountain ranges nearly devoid of sagebrush habitat, the Jackson Hole and Gros Ventre populations may have long been isolated from other Greater Sage-Grouse populations; however, only a few alleles were unique to the Jackson Hole and Gros Ventre populations. The observed genetic differentiation was largely due to allele frequency differences rather than private alleles, suggesting some historical gene flow. More work is needed to determine the timing of isolation and whether managers should focus on maintaining and increasing adequate sagebrush habitat, allowing the population to increase in size, or population supplementation to increase genetic diversity.