Fungal root endophytes colonize root tissue concomitantly with mycorrhizal fungi, but their identities and host preferences are largely unknown. We cultured fungal endophytes from surface-sterilized Cenococcum geophilum ectomycorrhizae of Betula papyrifera, Abies balsamea, and Picea glauca from two boreal sites in eastern Canada. Isolates were initially grouped on the basis of cultural morphology and then identified by internal transcribed spacer ribosomal DNA sequencing or by PCR restriction fragment length polymorphism. Phylogenetic analysis of the sequence data revealed 31 distinct phylotypes among the isolates, comprising mainly members of the ascomycete families Helotiaceae, Dermateaceae, Myxotrichaceae, and Hyaloscyphaceae, although other fungi were also isolated. Multivariate analyses indicate a clear separation among the endophyte communities colonizing each host tree species. Some phylotypes were evenly distributed across the roots of all three host species, some were found preferentially on particular hosts, and others were isolated from single hosts only. The results indicate that fungal root endophytes of boreal trees are not randomly distributed, but instead form relatively distinct assemblages on different host tree species.