Pinewood nematode (Bursaphelenchus xylophilus) is an invasive species that causes a destructive forest disease-pine wilt disease. This disease has been prevalent in some countries in Asia since the 1970s. An amplified fragment length polymorphism survey was used to compare the genetic variation of native and invasive nematode populations in China and to examine the changes in genetic diversity during the invasion process. The genetic diversity of Chinese populations was slightly higher than that of American populations. Analysis of groups sampled from different invasive stages in China, showed that no obvious change in genetic diversity. Hence, genetic drift and founder effects are not obvious in the invasion process. Phylogenetic analysis showed that Chinese pinewood nematode populations were closer to Japanese populations than to American populations. On the basis of the genetic relationships among samples, two major invasion pathways in China are suggested. One is from Guangdong to Anhui and Zhejiang, and the other is from Guangdong to Jiangsu and then from Jiangsu to Hubei, Guizhong and Congqing. The results imply that it is important to reinforce both domestic and international quarantine to control the spread of pinewood nematode.