Abstract: Enshi Prefecture in Hubei Province features a complex and diverse ecological environment. To investigate the distribution of macro-fungi resources, field surveys and transect sampling were conducted across eight counties and cities, including four national nature reserves. Through morphological and molecular biological studies, a total of 485 macro-fungal species were identified, belonging to 2 phyla, 8 classes, 21 orders, 83 families and 203 genera. Enshi City, Hefeng County and Lichuan City exhibited the highest species richness and the highest Shannon diversity index, indicating a high level of biodiversity. Dominant families included Russulaceae, Polyporaceae, Boletaceae, Amanitaceae and Agaricaceae. Dominant genera comprised Russula, Amanita, Entoloma, Gymnopus and Suillus. And dominant species were Candolleomyces candolleanus, Gymnopus dryophilus, Suillus bovinus, Trametes hirsuta and Lycoperdon perlatum. A total of 194 economically valuable macro-fungi were identified, including 2 near-threatened species and 15 endemic to China. A three-dimensional relationship exists between collection month, economic value and ecological type for macro-fungi, with a significant correlation between collection month and economic value for specific species. Mixed coniferous-broadleaf forests and deciduous broadleaf forests harbor the richest fungal species diversity among vegetation types. Edible and toxic fungi predominantly occur in mixed coniferous-broadleaf forests, while insect-parasitic fungi are exclusively found in mixed coniferous-broadleaf forests and coprophytic fungi only in deciduous broadleaf forests. Four new records for China were identified: Entoloma ammophilum, Psathyrella abieticola, Pseudosperma ushae and Hydropus omphaliiformis, enriching China's macro-fungal diversity. This study clarifies the diversity of macro-fungal resources in Enshi Prefecture, revealing a high proportion of edible and medicinal species and many species suitable for development and utilization. It also elucidates the relationships between macro-fungi and their environments, providing baseline data and a scientific basis for fungal conservation and subsequent resource development and utilization.