In 2005, researchers at the Leipzig Canopy Crane Research Facility collected living leaves of four temperate tree species at heights of between 15 and 33 m above the ground. Following surface sterilisation of the leaves, leaf-fragments were cultured on malt extract agar which allowed the growth of endophytic fungi into the surrounding medium. Isolated cultures were identified by morphology and sequence analysis of the D1/D2 region of the large subunit rDNA. Phylogenetic analysis established the taxonomic positions of the fungi. A total of 49 different taxa were identified, representing 20 families and ten orders. With the exception of one basidiomycetous yeast, all taxa belonged to filamentous ascomycetes. Species richness was highest on Tilia cordata and lowest on Quercus robur. Species-accumulation curves showed that the sampling effort was not sufficient to cover the majority of the likely species at the investigation site. Most endophytes proved to be ubiquitous within the canopy of the investigation site, but habitat preferences in terms of different tree species, different light regimes and season (sampling times) were obvious for some abundant endophytes. Apiognomonia errabunda and Aspergillus niger occurred predominantly on Q. robur, Diplodina acerina on Acer pseudoplatanus, one species of Phoma significantly prefered shaded leaves from the lower canopy layer whereas Sordaria fimicola prefered sun-exposed leaves from the upper tree crowns. Seasonal patterns were observed, for example, for A. errabunda, which was abundant in young leaves in the spring and almost completely absent in aged autumn-leaves, thus suggesting the accumulation of antifungal secondary plant metabolites during the growing season.