A history of timber harvest in West Virginia has reduced red spruce (Picea rubens) forests to < 10% of their historic range and resulted in considerable habitat fragmentation for wildlife species associated with these forests. The Virginia northern flying squirrel (Glaucomys sabrinus fuscus) has been described as a red spruce obligate subspecies that must traverse this fragmented landscape to disperse among remnant red spruce patches. Food availability in the forest matrix surrounding red spruce may be a limiting factor to successful dispersal of G. s. fuscus. We examined the diet of flying squirrels using stable isotope analysis and used vegetation surveys to determine the availability of diet items in the habitats encountered by G. s. fuscus in the matrix surrounding red spruce fragments. Stable isotope analysis suggested hypogeous fungi, epigeous fungi, and invertebrates contributed the most to the diet of G. s. fuscus, followed by lichen. Tree buds contributed the least in spring, and beechnuts contributed the least in fall. The vegetation surveys revealed that no habitat type had a greater availability of the diet items that contributed most to the assimilated diet of G. s. fuscus, suggesting that stand age and structure may be more important for diet-item availability than habitat type.