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Abstract In a study of fungi growing in various root-associated habitats in and around Picea mariana, black spruce, in northern Ontario, Canada, an examination was made of the degree to which differences in growth sites within an area of a few square kilometers might influence the structure of root-associated filamentous microfungal populations. Picea mariana roots were collected at four strongly differing boreal forest sites: an undisturbed forest site with deep litter and humus layers; a recently regenerated forest; a clearcut, former portable sawmill site with a few small, naturally regenerated trees; and an open peat bog penetrated by roots from trees growing along the margin. Comparisons were done on isolate assemblages primarily from serially washed mycorrhizae, supplemented with comparison samples from washed root bark and adherent rhizosphere soil. The Bray & Curtis similarity index and nodal components analysis were utilised to identify trends within the data. Root endophyte fungi, mainly Phialocephala fortinii and Meliniomyces variabilis, were among the most common isolates from serially washed mycorrhizae and showed strong trends among the site types, with the former most common from sites low in humus and also low in known humus-associated microfungi, and the latter most common from the peat bog site. The overall composition of the isolate assemblages from washed mycorrhizae mainly reflected site factors, with assemblages from the undisturbed and regenerated forest sites similar to one another and those from the clearcut and peat bog sites strongly distinct. A major difference was also seen between two seasonal samples at the exposed clearcut site, but few seasonal differences were seen at the other sites. The regenerated and undisturbed forest sites were high in Umbelopsis isabellina, Mortierella verticillata and Penicillium spinulosum, fungi typical of humic horizons in boreal podzols; the clearcut yielded the greatest numbers of Fusarium proliferatum, Umbelopsis nana and Penicillium montanense isolates, an assemblage tending to indicate exposed mineral soil; while the peat bog was typified by the presence of characteristic northern peat inhabitants Mortierella pulchella and P. spinulosum, as well as temperate peat inhabitant Penicillium lividum. A synthesis of these results with other data suggests that as a microhabitat, the mycorrhizosphere, as originally defined by Foster & Marks, is of little significance in determining the structure of filamentous fungal populations in soil influenced by the presence of ectomycorrhizal forest tree roots. Edaphic and overall microbial community conditions are much more significant, but the influence of a “symbiorhizosphere effect” exerted by certain ectomycorrhizal symbionts within the whole soil volume they occupy is also known in some cases and worthy of further investigation.