Understanding ectomycorrhizal fungal (EMF) community structure is limited by a lack of taxonomic resolution and autecological information. Rhizopogon vesiculosus and Rhizopogon vinicolor (Basidiomycota) are morphologically and genetically related species. They are dominant members of interior Douglas-fir (Pseudotsuga menziesii var. glauca) EMF communities, but mechanisms leading to their coexistence are unknown. We investigated the microsite associations and foraging strategy of individual R. vesiculosus and R. vinicolor genets. Mycelia spatial patterns, pervasiveness and root colonization patterns of fungal genets were compared between Rhizopogon species and between xeric and mesic soil moisture regimes. Rhizopogon spp. mycelia were systematically excavated from the soil and identified using microsatellite DNA markers. Rhizopogon vesiculosus mycelia occurred at greater depth, were more spatially pervasive, and colonized more tree roots than R. vinicolor mycelia. Both species were frequently encountered in organic layers and between the interface of organic and mineral horizons. They were particularly abundant within microsites associated with soil moisture retention. The occurrence of R. vesiculosus shifted in the presence of R. vinicolor towards mineral soil horizons, where R. vinicolor was mostly absent. This suggests that competition and foraging strategy may contribute towards the vertical partitioning observed between these species. Rhizopogon vesiculosus and R. vinicolor mycelia systems occurred at greater mean depths and were more pervasive in mesic plots compared with xeric plots. The spatial continuity and number of trees colonized by genets of each species did not significantly differ between soil moisture regimes.