In northern Europe, the biologically rich European aspen (Populus tremula) tends to disappear both from intensively managed and strictly protected forests, thus representing a biodiversity conservation challenge. This study describes the distribution of Aphyllophoroid fungi inhabiting aspen in a hemiboreal mixed-forest landscape where clear-cutting was usually followed by natural regeneration. In a 4-km2 area in Estonia, 181 individual forest stands and cut areas were surveyed. Sixty-one percent of the 2831 live aspen trees found were concentrated in 11% of the area. Thirty-six polypore species were recorded on aspen, with old stands and naturally created deadwood hosting most species. Among the records of 14 pre-defined aspen-specific basidiomycetes, Phellinus tremulae comprised about 95%. Six species (Ceriporiopsis aneirina, Clavicorona pyxidata, Funalia trogii, Phellinus populicola, Rigidoporus corticola, Steccherinum pseudozilingianum) were infrequent to rare, but widely distributed, inhabiting various stand-age classes, and mostly occurring outside “key habitats”. All the seven species absent are of conservation concern and frequent in Estonian old-growth forests. Thus, rotational management with natural aspen regeneration only provided habitat for some aspen-specific species. For sustaining all species, such landscapes should probably be complemented with stands managed using modified thinning and partial harvesting techniques in aspen-containing mixed stands.