Population structure, connectivity, and dispersal success of individuals can be challenging to demonstrate for solitary carnivores with low population densities. Though the cougar (Puma concolor) is widely distributed throughout North America and is capable of dispersing long distances, populations can be geographically structured and genetic isolation has been documented in some small populations. We described genetic structure and explored the relationship between landscape resistance and genetic variation in cougars in Washington and southern British Columbia using allele frequencies of 17 microsatellite loci for felids. We evaluated population structure of cougars using the Geneland clustering algorithm and spatial principal components analysis. We then used Circuitscape to estimate the landscape resistance between pairs of individuals based on rescaled GIS layers for forest canopy cover, elevation, human population density and highways. We quantified the effect of landscape resistance on genetic distance using multiple regression on distance matrices and boosted regression tree analysis. Cluster analysis identified four populations in the study area. Multiple regression on distance matrices and boosted regression tree models indicated that only forest canopy cover and geographic distance between individuals had an effect on genetic distance. The boundaries between genetic clusters largely corresponded with breaks in forest cover, showing agreement between population structure and genetic gradient analyses. Our data indicate that forest cover promotes gene flow for cougars in the Pacific Northwest, which provides insight managers can use to preserve or enhance genetic connectivity.