Aquatic fungi serve as core ecological engines in freshwater ecosystems, driving organic matter decomposition and energy flow to sustain environmental balance. Wetlands, with their distinct hydrological dynamics and nutrient-rich matrices, serve as critical habitats for these microorganisms. As an internationally designated Ramsar Site, Yunnan Dashanbao Black-Necked Crane National Nature Reserve in China not only sustains endangered black-necked cranes but also harbors a cryptic reservoir of aquatic fungi within its peat marshes and alpine lakes. This study employed high-throughput sequencing to characterize fungal diversity and community structure across 12 understudied wetland sites in the reserve, while analyzing key environmental parameters (dissolved oxygen, pH, total nitrogen, and total phosphorus). A total of 5829 fungal operational taxonomic units (OTUs) spanning 649 genera and 15 phyla were identified, with Tausonia (4.17%) and Cladosporium (1.89%) as dominant genera. Environmental correlations revealed 19 genera significantly linked to abiotic factors. FUNGuild functional profiling highlighted saprotrophs (organic decomposers) and pathogens as predominant trophic guilds. Saprotrophs exhibited strong associations with pH, total nitrogen, and phosphorus, whereas pathogens correlated primarily with pH. These findings unveil the hidden diversity and ecological roles of aquatic fungi in alpine wetlands, emphasizing their sensitivity to environmental gradients. By establishing baseline data on fungal community dynamics, this work advances the understanding of wetland microbial ecology and informs conservation strategies for Ramsar sites.