The geography of the Black Hills region of South Dakota and Wyoming may limit connectivity for many species. For species with large energetic demands and large home ranges or species at low densities this can create viability concerns. Carnivores in this region, such as cougars (Puma concolor), have the additive effect of natural and human-induced mortality; this may act to decrease long-term viability. In this study we set out to explore genetic diversity among cougar populations in the Black Hills and surrounding areas. Specifically, our objectives were to first compare genetic variation and effective number of breeders of cougars in the Black Hills during three harvest regimes: pre (2003-2006), moderate (2007-2010), and heavy (2011-2013), to determine if harvest impacted genetic variation. Second, we compared genetic structure of the Black Hills cougar population with cougar populations in neighboring eastern Wyoming and North Dakota. Using 20 microsatellite loci, we conducted genetic analysis on DNA samples from cougars in the Black Hills (n = 675), North Dakota (n = 113), and eastern Wyoming (n = 62) collected from 2001-2013. Here we report that the Black Hills cougar population maintained genetic variation over the three time periods. Our substructure analysis suggests that the maintenance of genetic variation was due to immigration from eastern Wyoming and possibly North Dakota.