Delineating wildlife population boundaries is important for effective population monitoring and management. The bobcat (Lynx rufus) is a highly mobile generalist carnivore that is ecologically and economically important. We sampled 1225 bobcats harvested in South Dakota, USA (2014–2019), of which 878 were retained to assess genetic diversity and infer population genetic structure using 17 microsatellite loci. We assigned individuals to genetic clusters (K) using spatial and nonspatial Bayesian clustering algorithms and quantified differentiation (F_ST and GST″) among clusters. We found support for population genetic structure at K = 2 and K = 4, with pairwise F_ST and GST″ values indicating weak to moderate differentiation, respectively, among clusters. For K = 2, eastern and western clusters aligned closely with historical bobcat management units and were consistent with a longitudinal suture zone for bobcats previously identified in the Great Plains. We did not observe patterns of population genetic structure aligning with major rivers or highways. Genetic divergence observed at K = 4 aligned roughly with ecoregion breaks and may be associated with environmental gradients, but additional sampling with more precise locational data may be necessary to validate these patterns. Our findings reveal that cryptic population structure may occur in highly mobile and broadly distributed generalist carnivores, highlighting the importance of considering population structure when establishing population monitoring programs or harvest regulations. Our study further demonstrates that for elusive furbearers, harvest can provide an efficient, broad-scale sampling approach for genetic population assessments.