Eleven polymorphic microsatellite DNA markers were screened to investigate patterns and processes of genetic variation in Allegheny woodrats at 2 spatial scales, geographically distinct populations and subpopulations within populations. The microsatellite markers detected considerable genetic variation and diversity, with an average heterozygosity of 62.0% (range 25.7–75.0%) and an average of 10.4 alleles per locus (range 5–19). Subpopulations conformed to Hardy–Weinberg expectations in 98.9% of comparisons, and allele frequency differed among subpopulations in 99.3% of comparisons. All statistical tests indicated significant genetic subdivision at the population and subpopulation levels. Theoretical estimates of gene flow were low among subpopulations, suggesting that effective dispersal is limited among subpopulations separated by as little as 3 km. A significant range-wide relationship between geographic and genetic distance at both scales suggests isolation by distance as a mechanism for the observed differentiation. The relationship between genetic distance and geographic distance among the 19 subpopulations within the central Appalachians was significant but weak. Results suggest that Allegheny woodrats associated with clusters of geographically proximate rock outcrops may function as population units. We recommend that individual rock outcrops or groups of geographically proximate outcrops be considered as population units for management.