Pine wilt is the most serious disease of native pines in Japan and potentially the most important nematode disease of conifers in the world. The pinewood nematode Bursaphelenchus xylophilus was found to be the causal agent. Difficulties arose with respect to the precise identity of some isolates of B. xylophilus and of similar species B. mucronatus and B. fraudulentus. Restriction enzyme analyses of repetitive DNA revealed bands specific for the species B. xylophilus, B. mucronatus and B. fraudulentus. Hybridization patterns obtained with unc-22 gene of C. elegans, clearly identified B. xylophilus, B. mucronatus and B. fraudulentus as well as the different geographic isolates of these species. Furthermore, it is possible to define the phylogenetic relationships between the different populations constituting the ‘pine wood nematode’ complex.