Despite being major drivers of the dynamics and diversity of plant populations and communities, the spatial variability of phyllosphere fungal assemblages has been seldom explored. We used CE-SSCP and 454 pyrosequencing to quantify the spatial variability of European beech phyllosphere fungal assemblages with a hierarchical sampling design including four different spatial scales (tree, branch, group of leaves and individual leaf). Fungal assemblages were highly diverse, with high proportions of generalist and cosmopolitan fungi. The major part of the variability was at the smallest spatial scale, between individual leaves. Within a single tree canopy, dissimilarity between fungal assemblages generally increased with distance between leaves. This pattern may be driven by within-canopy gradients of leaf traits and microclimate. At the stand scale, dissimilarity between fungal assemblages was correlated with the genetic distance rather than the geographic distance between trees, consistent with the findings of community genetics studies. Our results were constant over a small simulated sequencing depth, providing opportunities for the design of large-scale studies addressing the relationship between the genetic variation of trees and the variation of associated phyllosphere fungal assemblages.