Dispersal limitation and environmental filtering are two primary processes involved in shaping microbial community structure. The pristine environmental and geographical relatively isolation of small lakes distributed in the Headwater Region of Yellow River (HRYR) offer a unique opportunity to test the relative roles of these two processes on fungal communities. Here, we investigated the fungal community in sediment samples from 10 lakes located in the HRYR using high-throughput sequencing. The results showed that the fungal community was dominated by Sordariomycetes, Leotiomycetes, Dothideomycetes, Pezizomycetes and Agaricomycetes. The results revealed that altitude, mean annual temperature, C/N ration, dissolve organic carbon and total nitrogen were the best predictors for shaping fungal community structure in these lakes. Significant spatial and environmental distance decay relationships in the fungal community were detected. The partial Mantel test indicated that the fungal community structure was significantly correlated with environmental distance but not with geographic distance. Overall, environmental filtering plays a more important role than dispersal limitation in fungal community structure at a local scale in such an pristine and isolated region.