We investigated the species richness and composition of ectomycorrhizal (EM) fungi colonizing Pinus sylvestris L. seedlings naturally regenerating in boreal forest, in three different microhabitats: on forest ground, on decaying stumps, and within moss layer on erratic boulders. We tested the hypothesis that habitat differences would affect the composition of the EM community of regenerating pine seedlings. In total, 16 EM species were detected, from which none occurred on seedlings growing in all three microhabitats. Piloderma croceum and Cenococcum geophilum were common for seedlings growing in forest ground and on boulders, while Tricholoma aestuans and Suillus luteus were shared between seedlings growing on forest ground and decaying stumps. EM species richness and composition were strikingly different between seedlings regenerating in different microhabitats. Results are discussed as a function of dispersal and niche differentiation of EM fungi.