Sporocarps of hypogeous mycorrhizal fungi (truffles) are the major food of northern flying squirrels (Glaucomys sabrinus). The two subspecies of northern flying squirrels that occur in the southern Appalachians, G. s. coloratus and G. s. fuscus, are endangered species which are primarily found in the ecotone between high-elevation spruce-fir and northern hardwood forests. Our objective was to determine the microhabitat and macrohabitat characteristics associated with the presence and abundance of truffles in suitable habitat for northern flying squirrels. We sampled for truffles in 24–26, 1-m² plots on each of 10 northern flying squirrel sites in North Carolina and measured micro- and macrohabitat characteristics associated with sample plots and sites. Elaphomyces granulatus was the most common species of truffle found (78.7%). Red spruce (Picea rubra) was significantly more likely to be one of the three closest trees to plots with truffles. Further, spruce was the most important species in plots with truffles, followed by beech (Fagus grandifolia), red oak (Quercus rubra) and yellow birch (Betula lutea), whereas the most important species in plots with no truffles were beech, followed by yellow birch, spruce and red oak. At the macrohabitat (site) level, spruce was the most important species in sites with high truffle production followed by beech and red oak, whereas the most important species in sites with low truffle production were beech, yellow birch, spruce and rhododendron (Rhododendron spp.). Significant variables entered into a linear regression model predicting the number of truffles in a site were the importance values of fir (Abies fraseri), spruce and silverbell (Halesia carolina). Our data suggest that spruce-fir or mixed spruce-fir/hardwood stands are important foraging sites for northern flying squirrels in the southern Appalachians.