We investigated the community structure of ectomycorrhizal fungi under varying overstory tree compositions in the southern mixed-wood boreal forest of Quebec. Sampling took place at two locations of differing postfire ages and nine 100-m^2 plots were sampled per location. The dominant overstory tree species in the plots were trembling aspen (Populus tremuloides Michx.), white birch (Betula papyrifera Marsh.) or white spruce [Picea glauca (Moench) Voss], and balsam fir [Abies balsamea (L.) Mill.]. Mycorrhizae were analyzed using morphological as well as molecular methods, employing fungal-specific primers to amplify ribosomal DNA for subsequent cloning and sequencing. A total of 1800 mycorrhizal root tips collected from the 18 plots were morphologically classified into 26 morphotypes, with Cenococcum geophilum dominating (36% of root tips). A second set of root tips, selected from the same 18 samples on which the morphological analysis was based, were analyzed using molecular methods. From this analysis, 576 cloned polymerase chain reaction products were screened by restriction fragment length polymorphism analysis and a total of 207 unique types were found. No one type dominated the system and 159 occurred only once. Sequence analysis of the types that occurred more than once revealed that Piloderma sp., Russula sp., Cortinarius sp., and Lactarius sp. were the most common mycorrhizae. The ectomycorrhizal fungal community structure revealed by the rDNA analysis differed from that observed using morphological methods. Canonical correspondence analyses of the sequenced restriction types and % overstory composition indicate that the distributions of ectomycorrhizal fungi are influenced by the relative proportions of host tree species. The distinct fungal assemblages found in the different plots supported by the different combinations of host tree species provides further support for the need to conserve stand diversity in the southern boreal forest.