Small, fragmented populations are at greater risk of extirpation due to reduced genetic diversity from inbreeding and genetic drift. These processes ultimately decrease individual fitness and reduce the ability of a population to adapt. Hill's thistle (Cirsium hillii) is classified as a threatened species throughout much of its range, primarily due to the destruction and fragmentation of its habitat. This study addresses the impact of population size and isolation on the genetic diversity of Hill's thistle. We used microsatellite markers to genotype plants collected from the Lower Peninsula of Michigan in 2001, and from the Lower Peninsula and Drummond Island in 2012, in order to assess genetic differentiation across time and space, as well as to investigate rates of inbreeding in isolated and nonisolated populations. Genetic differentiation between the sample sites in the mainland population of Hill's thistle increased significantly between 2001 and 2012, indicative of increased fragmentation and isolation of the sample sites. However, the 2012 population exhibited lower inbreeding and no difference in heterozygosity or allelic diversity compared to 2001, suggesting the population is stable, or growing, in spite of isolation. Conversely, the population on Drummond Island displayed higher levels of inbreeding and lower number of effective alleles and heterozygosity compared to the mainland, typical of a small, isolated population. Our results indicate that the mainland population of Hill's thistle is persisting but should continue to be monitored demographically due to ongoing habitat loss.