Bursaphelenchus fungivorus is reported for the first time in Portugal, identified as associated with Pinus pinaster bark and characterized on the basis of morphological and morphometrical characters for this species. Species identification was confirmed using restriction fragment length polymorphism analysis and sequencing of the internal transcribed spacer (ITS) regions of ribosomal DNA. Intraisolate genetic variability was detected among ITS sequences of the Portuguese B. fungivorus isolate. Phylogenetic analysis, obtained from multiple sequence alignment between ITS sequences of Bursaphelenchus species, revealed that the Portuguese B. fungivorus isolate clusters with other B. fungivorus isolates, forming a separate group close to B. seani highlighting a molecular proximity of these two species.