Abstract: The paper aims to reveal characteristics of ectomycorrhizal fungi diversity in Quercus acutissima forest in subtropical montane, the sample plot of Q. acutissima natural forest community in mountainous area of central Guizhou was selected, and the diversity of EMF was analyzed by both morphological and molecular techniques. The findings indicated that a total of 27 OTUs of EMF were identified, belonging to 2 phyla, 4 classes, 8 orders, 9 families and 10 genera, of which the Basidiomycota and Ascomycota accounted for 86.05% and 13.95%, respectively. Indicating that the richness of Basidiomycota was significantly higher than that of Ascomycota. Russulaceae and Thelephoraceae were the dominant EMF groups. Russula and Tomentella were the dominant genera. The richness of Xerocomus, Hebeloma, Thelephora, Laccaria and Tuber were the least.