A polymerase chain reaction(PCR) was used to amplify ribosomal DNA containing 5.8 S gene and internal transcribed spacer region 1(ITS1). According to the difference between the sequences of internal transcribed spacer region 1(ITS1) of Bursaphelenchus xylophilus and B.mucronatus, the species of specific primers were designed respectively for the two species nematodes. A single nematode, living or preserved in formalin, could be detected rapidly, and B.xylophilus and B.mucronatus were distinguished as well. This method would be useful for rapid detection of nematodes.