The Western Prairie Fringed Orchid (WPFO) Platanthera praeclara is listed as a federally threatened species in the United States. The habitat of the WPFO is highly fragmented, causing the possibility of genetic isolation among populations. I assessed microsatellite polymorphism in eight WPFO populations using six polymorphic microsatellite markers. High levels of within population genetic diversity were found: 12.5 alleles per locus and mean expected heterozygosity (HE) of 0.622. The genetic structure among the populations was weak; the overall FST value was 0.0692. A mantel test showed no correlation between genetic and geographic distance r = 0.2793 (P = 0.31). The genetic diversity and genetic structure is comparable to other plant species. Significant inbreeding many be occurring as indicated by a FIS value of 0.1903. More studies with the developed markers are necessary over a larger part of the geographic range of WPFOs to determine if other populations are genetically isolated.