Below- and above-ground microbial communities are expected to dramatically change following the loss of healthy Tsuga canadensis (Eastern Hemlock) ecosystems due to the effects of Adelges tsugae (Hemlock Woolly Adelgid [HWA]) infestation. In 2006, few healthy Eastern Hemlock stands remained in Great Smoky Mountains National Park (GRSM), and we sought to obtain baseline soil microbial data from these undisturbed areas to better understand them. This study surveyed root fungal communities (e.g., mycorrhizae) using molecular cloning and cultural methods at 2 remaining healthy Eastern Hemlock stands in GRSM. We sampled roots from 40 trees representing 2 age classes (<75 and >150 years old) and 2 elevations (486 m and 1158 m) to obtain molecular clones and isolated cultures. Molecular clones from the ITS region indicated the presence of 124 taxa in 23 genera. The dominant genera were Piloderma (8.2%), Russula (6.8%), Tomentella (4.0%), Meliniomyces (3.4%), and Clavulina (2.9%). Ninety-seven of the taxa identified occurred at <1.0% relative abundance. The most frequent fungal species were Meliniomyces variabilis (22.6%), Russula granulata (16.1%), Phialocephala fortinii (9.7%), and Cladophialophora chaetospira (16.1%). Species richness, diversity, and evenness for total fungi, ectomycorrhizal species, and saprobic species were similar at different elevations and stand ages. Using ITS sequence data, we identified 9 species from 1800 fungal isolates from Eastern Hemlock root tissue, the most common of which were Trichoderma harzianum (24.4%), Trichoderma viride (24.4%), Trichoderma asperellum (17.1%), and Ilyonectria radicicola (12.2%). Species richness, diversity, and evenness were not significantly different across locations for the molecular data. Baseline data for the fungal taxa identified in the study may be important for understanding ecological changes that can occur post-HWA damage and HWA-caused stand losses. These data could also be used to support Eastern Hemlock reforestation efforts within current or former habitats in GRSM and surrounding ecosystems.