The endophytic fungi in the roots of Rheum lhasaense at 4 200 m and 5 100 m are analyzed using high-throughput sequencing technology to explore their diversity.The results show that there was no significant difference in the abundance and evenness of endophytic fungi at the two altitudes.Ascomycota and Basidiomycota are the top two phyla,while Penicillium,Ilyonectria and Cladosporium are the top three genera at 4 200 m;Penicillium,Dactylonectria,and Plectosphaerella are the top three genera at 5 100 m.These findings provide a theoretical basis for further exploring how Rheum lhasaense adapts to cold and high altitude environments as well as developing its medicinal resources from endophytic fungi.