Phlox pungens is a rare taxon restricted to cold deserts in the Wyoming Basins Ecoregion of west-central Wyoming, with occurrences in both the Wind River Basin (from which it was originally described) and the Green River Basin (populations that have been characterized as a different “morph”). The population genetics of P. pungens was investigated using microsatellite markers developed for the genus. Four microsatellite loci were genotyped from a total of 200 individuals of P. pungens from 10 sites. Ploidy levels were also assessed using flow cytometry. Most sampled populations of P. pungens were tetraploid, but 2 populations in the Green River Basin were diploid, and one high-ploidy population was discovered in the Wind River Basin. Mitotic chromosome counts were conducted for the high-ploidy site and documented the presence of 10x individuals (2n = approximately 70). Microsatellite data analysis revealed moderate genetic differentiation between populations of P. pungens, notably structured by geography with the Wind River Basin versus the Green River Basin (i.e., the 2 morphs) corresponding closely to different genetic groups. Cytotypic variation generally did not impact genetic patterns, with the exception of the high-ploidy population, which constituted a third genetic group with a high number of alleles per locus and many private alleles. The genetic differences between the 2 morphs were driven more by different allele frequencies than by fixation for different alleles at these microsatellite loci. The results of this study highlight the genetic differentiation between populations of P. pungens in the Wind River versus the Green River Basins. Moreover, P. pungens is part of a taxonomically challenging set of glandular cespitose Phlox taxa distributed in western North America, and our findings on population genetic variation in this rare taxon contribute to ongoing work on taxonomy and diversity in the Phlox study system.