ABSTRACT The ectomycorrhizal fungi associated with American chestnut (Castanea dentata) were characterized using DNA extracted from the root-tips of naturally occurring saplings from a forest in northeastern Tennessee. A total of 18 taxa were recorded, although one of these (Oidiodendron maius) was surprising because it has been reported previously to form what appear to be mycorrhizal associations only with members of the Ericaceae. One-third of the recorded taxa were representatives of the genera Russula or Lactarius (both members of the family Russulaceae), and three other genera (Cortinarius, Tomentella, and Tricholoma, each represented by two species). The data presented herein at least suggest that members of the Russulaceae are among the most common and widespread ectomycorrhizal fungi associated with naturally occurring individuals of American chestnut in the forests of eastern North America in which it was once dominant. However, it should be pointed out that our data are limited and thus are best considered as preliminary.