Microsatellite markers are valuable tools for determining amount and distribution of genetic diversity and differentiation within and between populations. In this study we examined the level of microsatellite variability within and among five populations of Lobelia villosa, a rare Hawaiian endemic lobeliad on the island of Kaua‘i. Populations of L. villosa were sampled from two regions on Kaua‘i: two populations at the Kilohana Lookout area and three populations from Alaka‘i Swamp. Nineteen microsatellite DNA primers were developed for L. villosa, 12 of which demonstrated polymorphism and were subsequently multiplexed and labeled for genotyping. An overall moderate degree of genetic differentiation was found within and between populations. Pairwise F_ST data showed population structure, and analysis with Structure software indicated two genetic clusters (K = 2) corresponding to the two sampled geographic regions. Although L. villosa exhibits moderate diversity, which exceeds that of other Hawaiian endemics with restricted distributions, measurements of F_IS were positive across 10 out of 12 loci, suggesting that inbreeding is occurring at the population level.