For diagnosis of pine wilt disease, a simple PCR-RFLP method was developed to identify and to differentiate two similar nematode species, based on a living or preserved single specimen. Pinewood nematodes, Bursaphelenchus xylophilus, and Bursaphelenchus mucronatus were examined. A single nematode in 1l of distilled water was put on a glass slide. When the water had almost dried the nematode was crushed with a filter paper chip, 1.5mmà1.5mm, with the aid of forceps. The filter paper chip containing nematode remains was immediately placed into PCR buffer as the DNA template. The primer set used was to amplify ribosomal DNA containing the inter-transcribed spacer (ITS) 1, 5.8S and ITS2 regions. The PCR product was consistently obtained from a single nematode, and digesting the product with restriction endonuclease, Hinf I, enabled discrimination between B. xylophilus and B. mucronatus. This method was simple, convenient and definitive, and could successfully determine the pathogen in the diagnosis of pine wilt disease. This method was applicable also to nematode specimens preserved under various conditions except in the case of those preserved in aldehyde-containing fixatives.