Fungi represent key ecosystem factors that affect plant growth and development and improve soil structure and fertility. Due to changes in environmental conditions, fungi show strong spatial heterogeneity along altitudinal gradients. Current knowledge of the driving mechanisms and effects of soil fungal community construction at high altitudes is very limited on a regional scale. We collected soil samples from alpine grasslands at six altitudinal gradients (2813–5228 m) in the high-altitude area of the Qinghai-Tibet Plateau. The horizontal distance of the sampling zone spanned 1500 km. Distribution patterns, key influencing factors for soil fungal diversity, and dominant mechanisms of ecological processes in the alpine grasslands were analyzed. We found that the diversity of the soil fungal communities was significantly different at different altitudes; with increasing altitude, the number of fungal species increased. Mucoromycota was better adapted to alpine grassland ecosystems at altitudes of above 4000 m. Dispersal limitation was the main ecological control process among stochastic processes. With the increase of altitude, the dominant role of dispersal limitation gradually decreased, and the proportion of other random processes such as ecological drift gradually increased. In this study, soil geochemical factors (soil organic carbon, SOC; total phosphorus, TP) mainly influenced the composition of the fungal community in the low altitude region, while climatic factors (mean annual temperature, MAT) were the key factors and main driving forces for the composition of the soil fungal community in the alpine meadow in the high altitude region. This study supplements the information on the biogeographic distribution patterns and environmental drivers of fungal communities along altitudinal gradients at high altitudes on a regional scale. Our results highlight the effects of temperature change on fungal community composition in high altitude regions of alpine grasslands. Subsurface fungal communities should be considered when predicting the function of alpine grassland ecosystems under future climate change.