Mating behavior and social structure can influence genetic structure within and between populations, yet most studies focus on highly kin-structured, polygynous species. The North American beaver (Castor canadensis) is socially monogamous; however, examination of recent genetic data suggests that this species may be opportunistically promiscuous. We used 7 microsatellite loci to quantify genetic structure within and between 2 beaver populations in Illinois. An analysis of molecular variance revealed significant genetic subdivision among breeding groups in southern Illinois (F_ST = 0.086, P < 0.001), whereas regional genetic subdivision was evident in central Illinois (F_ST = 0.037, P < 0.001). Overall F_ST between populations also was significant (0.068 ± 0.012 SE, P < 0.001). Bayesian clustering assigned individuals from the 2 geographic sampling regions into 2 distinct genetic clusters with 70% of individuals assigned to 1 of the 2 clusters. Migration between populations was low at 0.16 individuals/generation (confidence interval = 0.0079–0.33). Estimates of population subdivision, cluster analysis, and dispersal indicate that these populations are genetically distinct, but are connected by infrequent dispersal.