The Agaricomycotina are a phylogenetically diverse group of fungi that includes both saprotrophic and mycorrhizal species, and that form species – rich communities in forest ecosystems. Most species are infrequently observed, and this hampers assessment of the role that environmental heterogeneity plays in determining local community composition and in driving β-diversity. We used a combination of phenetic (TRFLP) and phylogenetic approaches [Unifrac and Net Relatedness Index (NRI)] to examine the compositional and phylogenetic similarity of Agaricomycotina communities in forest floor and surface soil of three widely distributed temperate upland forest ecosystems (one, xeric oak – dominated and two, mesic sugar maple dominated). Generally, forest floor and soil communities had similar phylogenetic diversity, but there was little overlap of species or evolutionary lineages between these two horizons. Forest floor communities were dominated by saprotrophic species, and were compositionally and phylogenetically similar in all three ecosystems. Mycorrhizal species represented 30% to 90% of soil community diversity, and these communities differed compositionally and phylogenetically between ecosystems. Estimates of NRI revealed significant phylogenetic clustering in both the forest floor and soil communities of only the xeric oak-dominated forest ecosystem, and may indicate that this ecosystem acts as a habitat filter. Our results suggest that environmental heterogeneity strongly influences the phylogenetic β-diversity of soil inhabiting Agaricomycotina communities, but has only a small influence on forest floor β-diversity. Moreover, our results suggest that the strength of community assembly processes, such as habitat filtering, may differ between temperate forest ecosystems.