Temperate ectomycorrhizal (ECM) fungi show segregation whereby some species dominate in organic layers and others favor mineral soils. Weak layering in tropical soils is hypothesized to decrease niche space and therefore reduce the diversity of ectomycorrhizal fungi. The Neotropical ECM tree Dicymbe corymbosa forms monodominant stands and has a distinct physiognomy with vertical crown development, adventitious roots and massive root mounds, leading to multi-stemmed trees with spatially segregated rooting environments: aerial litter caches, aerial decayed wood, organic root mounds and mineral soil. We hypothesized that these microhabitats host distinct fungal assemblages and therefore promote diversity. To test our hypothesis, we sampled D. corymbosa ectomycorrhizal root tips from the four microhabitats and analyzed community composition based on pyrosequencing of fungal internal transcribed spacer (ITS) barcode markers. Several dominant fungi were ubiquitous but analyses nonetheless suggested that communities in mineral soil samples were statistically distinct from communities in organic microhabitats. These data indicate that distinctive rooting zones of D. corymbosa contribute to spatial segregation of the fungal community and likely enhance fungal diversity.