Shifts in ectomycorrhizal (ECM) community structure were examined across an experimental hydrologic gradient on containerized seedlings of two oak species, Quercus montana and Quercus palustris, inoculated from a homogenate of roots from mature oak trees. At the end of one growing season, seedlings were harvested, roots were sorted by morphotype, and proportional colonization of each type was determined. DNA was subsequently extracted from individual root tips for polymerase chain reaction, restriction fragment length polymorphism, and rDNA sequencing of the ITS1/5.8S/ITS2 region to determine identities of fungal morphotypes. Twelve distinct molecular types were identified. Analysis of similarity showed that ECM fungal assemblages shifted significantly in composition across the soil moisture gradient. Taxa within the genus Tuber and the family Thelephoraceae were largely responsible for the changes in fungal assemblages. There were also significant differences in ECM community assemblages between the two oak host species. These results demonstrate that the structure of ECM fungal communities depends on both the abiotic and biotic environments and can shift with changes in soil moisture as well as host plant, even within the same genus.