Abstract: Bursaphelenchus xylophilus, B. mucronatus and the other nematodes separated from dead pine. were detected by using primer K09F2/R. The results indicated that the PCR product of all B. xylophilus isolates had a clear and bright fragment about 340bp. But B. mucronatus and other nematodes had not any fragments. So pairwise K09 F2/R was used for constructing identification kit of B. xylophilus. It took a short time to identify nematodes by adopting this kit. The time of total detection process was within two and half hours. Besides, the sensitivity of this kit reached to 1/7 of a nematode. The detection result is easy to estimate, and the cost of detection is quite low. This kit is a rapid, reliable and convenient means for detecting B. xylophilus.