Abstract: In this study, the hybridization was conducted between four Bursaphelenchus xylophilus strains selected from China, Japan, the United States and two B. mucronatus strains selected from China. The results showed that, hybridization could occur between some B. xylophilus strains and B. mucronatus strains, the success rate of negative crossing was two times that of positive crossing,but hybridization rate was extremely low, and the hybrid offspring was difficult to form a sustainable breeding population. The F1 hybrids larvae contained both Bx molecular markers and Bm molecular markers by using Bx/Bm quick molecular detection system. Inter-specific hybrids showed different gene types after subculturing 30 days, the offspring nematodes of BxAN5♀×Bm JNL1 ♂,BxAN5 ♂×BmAN5♀ and BxUSA ♂×Bm JNL1♀ tended to become the populations which dominant species was Bx, as well as the offspring of BxJNL5 ♂×BmJNL1♀ tended to become the population which dominant species was Bm. If intraspecific matings could happen, both B. mucronatus and B. xylophilus were inclined to have intraspecific mating priority. The adaptability of B. xylophilus in mating was better. There was little possibility of reproductive isolation between Bx and Bm which geographically close, and their inter-specific hybrids were easily to produce. The reproductive isolation existed between B. mucronatus and B. xylophilus, and introgression might happen but hybrids were difficult to form a sustainable reproductive offspring.