Knowledge on the diversity of hypogeous sequestrate ascomycetes is still limited in the Balkan Peninsula. A new species of truffle, Tuber pulchrosporum, is described from Greece and Bulgaria. Specimens were collected from habitats dominated by various oak species (i.e. Quercus ilex, Q. coccifera, Q. robur) and other angiosperms. They are morphologically characterised by subglobose, ovoid to irregularly lobed, yellowish-brown to dark brown ascomata, usually with a shallow basal cavity and surface with fissures and small, dense, almost flat, trihedral to polyhedral warts. Ascospores are ellipsoid to subfusiform, uniquely ornamented, crested to incompletely reticulate and are produced in (1–)2–8-spored asci. Hair-like, hyaline to light yellow hyphae protrude from the peridium surface. According to the outcome of ITS rDNA sequence analysis, this species forms a distinct well-supported group in the Aestivum clade, with T. panniferum being the closest phylogenetic taxon.