We used a culture-dependent approach to investigate root-associated fungal communities in Fagaceae roots at four fagaceous species-dominant forests in Japan. In total 1029 isolates were collected and classified, based on colony morphological features and molecular information. Species of order Helotiales (Ascomycota) were dominantly isolated at all four sites, in which a globally-distributed putative endophytic group in Hyaloscyphaceae predominated. This group of fungi was morphologically and phylogenetically investigated using these isolates as well as additional isolates collected from 8 different sites in Japan. Among the Hyaloscyphaceae, Glutinomyces species were frequently detected, and three novel species, G. inflatus, G. vulgaris, and G. takaragaikensis spp. nov. were identified and described according to their morphology and genealogical concordance.