Ectomycorrhizal (ECM) associations are fundamental for seedling establishment in many forest ecosystems. However, it is unclear how spatial and environmental factors affect the structure of ECM fungal communities at the seedling establishment stage. Here, we investigated how spatial and environmental factors affect the composition of ECM fungal communities on naturally established seedlings of Pinus pumila in nine ice-age relict forests and compared the results with those of mature shrubs that we previously characterized. ECM fungi on field-collected seedling roots were identified using sequencing of the internal transcribed spacer (ITS). Redundancy analysis revealed that climatic factors accounted for the largest variation in ECM fungal community composition in naturally established seedlings (40%). The difference in host stages (seedlings/mature trees) in ECM fungal communities was larger than those of spatial, soil, and climate factors at the regional scale. Our results indicate that the ECM fungi that facilitate seedling establishment are significantly different from those on mature trees and are independently affected by climatic factors at the regional scale.