We investigated nest site use of the federally endangered northern flying squirrel (Glaucomys sabrinus coloratus) in southwest Virginia at Mount Rogers National Recreation Area. In second-growth and old forests, number of nests used by both sexes combined was x̄ = 3.3 ± 0.8 (females n = 3, x̄ = 2.7 ± 0.9, males n = 8, x̄ = 3.7 ± 1.2, overall range = 1–6). Nest site characteristics varied and flying squirrels used a variety of nest types including subterranean structures. We found northern flying squirrels chose larger taller nest trees regardless of nest tree type [red spruce (Picea rubens), n = 3, P = 0.04, deciduous, n = 8, P = 0.02, snags, n = 3, P = 0.01] compared to the available trees of the same type within a 200 m² area of the nest tree. Nest sites in old forest stands (i.e., ≥125 y old) were characterized by greater downed wood (% cover/ha) that can be associated with the increased density of fungal colonies, lower snag density (n-snags/ha) that means a more open midstory for clearer glide paths, lower live-tree density (n-trees/ha) relating to clearer glide paths and lower shrub cover (% cover/ha) indicating a more open understory compared to nest sites in second-growth stands.