Pine wood wilt disease caused by the pine wood nematode, Bursaphelenchus xylophilus , has been a serious problem in the southern regions of Korea. Efficient diagnosis of B. xylophilus from infected pine wood specimens is critical for the management of this pest. Traditional microscopic examination often results in an erroneous identification because a closely related non-pathogenic species, B. mucronatus, has a great degree of morphological similarity to B. xylophilus. In an attempt to search for reliable molecular markers for the discrimination of these species, we have cloned the 5S rRNA genomic DNA fragments containing both coding and intergenic spacer (IGS) regions from B. xylophilus and B. mucronatus through a homology-probing PCR strategy. Sequence analyses revealed that coding sequences of the 5S rRNA gene from the two species are almost identical (98.3% homology) but that the IGS sequences differ substantially between the species. Based on the IGS sequence differences (69.7% homology), we designed species-specific primer sets and developed a PCR-based diagnosis protocol for the identification and discrimination of the two nematode species on a molecular basis.