The availability and the quality of substrates are important drivers of macrofungal biogeography, and thus macrofungal species occurrence is potentially dependent on the availability of different substrates. However, few studies have explored the properties of macrofungal substrates and assessed the relationship between macrofungal diversity and substrate diversity at a landscape level. To address this issue, we conducted a landscape-scale survey of basidiocarp substrates in the Greater Mekong Subregion (GMS). A total of 957 macrofungal species distributed across 73 families and 189 genera were collected. Substrates of these macrofungi were categorized into four main groups (namely, litter, soil, root, and rare substrates) and referenced into 14 sub-substrate types (such as branches, leaves, and fruit). The results revealed that 50% of the observed macrofungal species were symbiotrophs living in ectomycorrhizal association with plant hosts, 30% were saprotrophs decomposing plant litter, 15% lived in soil organic matter, and 5% lived in rare substrates. The most abundant root symbiotic fungi were members of Russula, whereas most litter saprotrophic fungi belonged to Marasmius. Macrofungi commonly favored a single substrate. This specificity was not affected by changes in vegetation or climate. Less than 1% of macrofungi (e.g., Marasmius aff. maximus) could live on multiple substrates. Most of these unusual macrofungi were characterized as highly mobile and were generally found in successional areas. In secondary forests, our survey indicated that significant correlations exist between substrate preference and taxonomic diversity, reflected as higher substrate diversity generally accompanied by higher macrofungal diversity. In conclusion, substrate preference is an important factor driving macrofungal composition and distribution in the GMS. Macrofungi that thrive on multiple substrates constitute pioneer groups that have an important role in establishing macrofungal communities in new habitats. These observations have furthered our understanding of how substrate preferences could explain macrofungal biogeography.