The pine wood nematode Bursaphelenchus xylophilus parasitizes millions of pine trees worldwide each year, causing severe wilt and the death of host trees. Glycoside hydrolase 45 genes of B. xylophilus are reported to have been acquired by horizontal gene transfer from fungi and are responsible for cell wall degradation during nematode infection. Previous studies ignored the possibility of copy number variations of such genes. In this study, we determined that two of the glycoside hydrolase 45 genes evolved to maintain multiple copies with distinct expression levels, enabling the nematode to infect a variety of pine hosts. Additionally, tandem repeat variations within coding regions were also detected between different copies of glycoside hydrolase 45 genes that could result in changes in protein sequences and serve as an effective biological marker to detect copy number variations among different B. xylophilus populations. Consequently, we were able to further identify the copy number variations of glycoside hydrolase 45 genes among B. xylophilus strains with different virulence. Our results provide new insights into the pathogenicity of B. xylophilus, provide a practical marker to genotype copy number variations and may aid in population classification.