The specificity of fungal endophytes to trees has generally been investigated on allopatric populations. In the present study, specificity was investigated on Quercus cerris and Quercus pubescens growing in sympatry at the same site, to examine host-endophyte interactions without interference from habitat-specific selective pressure. In a two-year study, 4800 samples were obtained from photosynthetic and non-photosynthetic tissue (leaves and twigs) both of healthy and declining trees. Endophytes were isolated from surface-sterilized samples and identified by traditional or molecular methods. Twenty-two endophyte species in 19 genera were identified. Some taxa in the Sordariomycetes, Dothideomycetes and Leotiomycetes colonized both species, but quantitative differences were evident. Water shortages and extended droughts may have impacted more severely on Q. cerris, which exhibited a more diverse endophytic assemblage and greater infection levels than Q. pubescens. In both species, more isolates were recovered from twigs than from leaves, and more from declining than from healthy trees. Endophytes tended to be specific to each host, and to the organs of that host. Interaction between plant species and the environment and continued competitive interaction between endophyte species may have led to niche diversification, with selection favouring host-specific and organ-specific endophytes. This study advances understanding of the role of some pathogenic fungal endophytes in Mediterranean oak forests.