The Eastern Massasauga (Sistrurus catenatus) is a small, grassland-dependent rattlesnake species declining throughout its native range, and is thus a species of high conservation priority. In Illinois, only a single population remains of a once widespread distribution. We documented genetic diversity in this population over a ten-year period and assessed levels of heterozygosity, allelic diversity, inbreeding (F_IS), and effective population size (N_e). Neither heterozygosity nor levels of inbreeding differed significantly among periods. We identified 21 alleles that occurred in a single time period, some of which may have been lost from the population given our estimated detection probability of 93%. Effective population size (N_e) was numerically small and showed a decreasing trend through time. Despite small population size and a lack of connectivity, there was no significant decline in genetic diversity over the ten-year study. Aspects of life history, coupled with a preference for a historically patchy habitat, may mitigate the loss of genetic diversity in the species and promote their persistence in the fragmented habitats of the Anthropocene. However, continued genetic monitoring is recommended, and population recovery measures should be implemented as soon as possible to mitigate the deleterious effects of small population size.