Fungi regulate key nutrient cycling processes in many forest ecosystems, but their diversity and distribution within and across ecosystems are poorly understood. Here, we examine the spatial distribution of fungi across a boreal and tropical ecosystem, focusing on ectomycorrhizal fungi. We analyzed fungal community composition across litter (organic horizons) and underlying soil horizons (0-20 cm) using 454 pyrosequencing and clone library sequencing. In both forests, we found significant clustering of fungal communities by site and soil horizons with analogous patterns detected by both sequencing technologies. Free-living saprotrophic fungi dominated the recently-shed leaf litter and ectomycorrhizal fungi dominated the underlying soil horizons. This vertical pattern of fungal segregation has also been found in temperate and European boreal forests, suggesting that these results apply broadly to ectomycorrhizal-dominated systems, including tropical rain forests. Since ectomycorrhizal and free-living saprotrophic fungi have different influences on soil carbon and nitrogen dynamics, information on the spatial distribution of these functional groups will improve our understanding of forest nutrient cycling.