Species of Heterobasidion are amongst the most destructive disease agents on conifers in the Northern hemisphere. The area examined in the present work was the first example of typical disease centres and symptoms of H. annosum root rot observed on pines in Turkey. Twenty disease centres were identified in a Pinus brutia regeneration area, and a total of 182 isolates were obtained from the centres. All isolates from the regeneration area mated almost perfectly with H. annosum s.s. isolates from Europe, giving a clear identification. Based on a combination of morphological and molecular methods, the genetic variation of H. annosum was determined in the study site. The variation amongst and within isolates amounted to, respectively, 49% and 51% of total variation. The population structure of the isolates is discussed. It was concluded that H. annosum was well‐established in the regeneration area and was probably not caused by recent infections and spread.