Allegheny woodrats (Neotoma magister) have been declining over the past 30 years, with the most precipitous declines occurring along the northern and western parts of their range. Although reduced genetic diversity has been implied as a possible reason for Allegheny woodrat declines, genetic analyses have been limited to localized regions within their range and no state-wide efforts have been undertaken. To determine the amounts and patterns of genetic variability and existing population structuring across Virginia, genetic analyses were performed on 157 Allegheny woodrats using 21 microsatellite loci and a 266 base pair sequence of the cytochrome b (cytb) gene of mitochondrial DNA (mtDNA). Genetic diversity of contemporary Allegheny woodrat samples was compared to diversity of 84 historic Virginia specimens from 1860 to 1989. Furthermore, to better understand ecological and evolutionary processes influencing gene flow and connectivity for Allegheny woodrats across the landscape, I used a landscape genetics approach to model the effects of features with the potential to affect Allegheny woodrat movement and gene flow including land cover, roads, rivers, and elevation. Mean heterozygosity across all populations was 62.0%, (range 21.5%-95.2%) with a mean of 8 alleles/locus, (range 4-18). Three populations were identified based on conformity to Hardy-Weinberg expectations. FST and GST values indicate moderate to great differentiation between populations, respectively. Contemporary and historic genetic diversity was compared using nested fragments (138bp; 266bp) of cytb and analyses of 21 microsatellite loci. Overall mtDNA haplotype diversity values were relatively high (0.760 for the 138bp sequence; 0.830 for the 266bp sequence), whereas nucleotide diversity values were low (0.009 for the 138bp sequence, 0.007 for the 266bp sequence), indicating that Allegheny woodrat haplotypes are closely related. Haplotype richness in the 266bp sequence was lower in historic (7) versus contemporary (20) samples. We identified 8 microsatellite alleles in historic specimens not present in contemporary samples. Modeling suggested that >50m wide rivers and lower elevations impede gene flow across the landscape. Higher elevations (750m and 500m) generally indicate more suitable Allegheny woodrat habitat. Management should focus on mimicking natural patterns of movement across the landscape by enhancing connectivity corridors or through artificial means.