Pine wilt disease, a devastating infectious disease of pine trees, is caused by the pinewood nematode, Bursaphelenchus xylophilus. It is important to understand the spatial dispersal processes of B. xylophilus within host-tree tissues to assess its pathogenic mechanism. However, this is not feasible with conventional microscopy-imaging techniques. In this study, we showed that X-ray micro-computed tomography (micro-CT) imaging can be a powerful tool for visualizing infected nematodes within host-tree tissues. We visualized 161 nematodes and 11 eggs within a Pinus thunbergii stem section, 47.3 mm³ in volume, using an appropriate segmentation of the micro-CT images. Quantitative measurements of the segmented region corresponding to the nematodes allowed for the calculation of the longitudinal length and a-value, which were similar to previous morphological descriptions of B. xylophilus. The technique adopted in this study can aid in understanding the behavior of and obtaining quantitative information on B. xylophilus within tree tissues.