Abstract: Xingshan County of Hubei Province is situated in the Three Gorges Reservoir Area. In order to know well macro-ascomycetous species in this county and improve the understanding of the biodiversity of the Three Gorges Reservoir Area, a series of survey of macro-ascomycetous and collecting of specimens were carried out in the duration from October of 2016 to August of 2018. The specimens were identified based on morphological and molecular systematic methods. A total of 96 species belonging to 4 classes, 5 orders, 20 families, and 48 genera were confirmed. Among them, forty species were recorded for the first time in Hubei Province, such as Ascocoryne cylichnium, Bisporella citrina, Chlorociboria poutoensis and so on. The most dominant genus was Peziza, including 9 species accounting for 9.38% of the total species, and the most dominant family was Pyronemataceae, including 17 species accounting for 17.71% of the total species. Most of the species were widely distributed in the areas with 800-1 600 m above sea level and mainly grown in the evergreen and deciduous broad-leaved mixed forest. Lachnum abnorme was found as the widely distributed species. There were 41 species of geophilous fungi, 40 species of lignicolous fungi, 10 species of entomogenous fungi, 2 species of fungicolous fungi and 3 species of geophilous or lignicolous fungi. A phylogenetic tree based on the LSU rDNA sequence was constructed, showing the relationship among major macro-ascomycetes species in Xingshan County.