【Objective】This study aims to identify suitable metabolites for the early diagnosis of pine wilt disease (PWD). To achieve this, the metabolome of stem segments from Pinus massoniana infected by different nematodes was analyzed, with the goal of improving the prevention and control of PWD.【Method】With four-year-old P. massoniana trees with similar growth characteristics were inoculated with three different nematode species, each with varying pathogenicity. These included the highly virulent Bursaphelenchus xylophilus (nematode strain FCBX), the highly virulent B. mucronatus (nematode strain BM7), and the non-virulent B. mucronatus (nematode strain FCBM). These nematodes were isolated and cultured in the Forest Pathology Laboratory of Nanjing Forestry University. Inoculation of P. massoniana trees with these nematodes were conducted under controlled conditions to assess their virulence. A sterile water control group (CK) was also included. Three days after injection, the metabolome of stem segments from P. massoniana was analyzed using ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Principal component analysis (PCA) was used to ensure quality control of the metabolomic data. Metabolites with specific expression following B. xylophilus inoculation were identified through a combination of P-values and variable importance in projection (VIP) scores. Targeted detection and validation of these metabolites were conducted using ultra-performance liquid chromatography (UPLC).【Result】The three nematode species exhibited significant differences in virulence toward P. massoniana. Metabolomic analysis of stem segments revealed reliable results. One key finding was that 7-dehydrocholesterol was a common differential metabolite across the comparative metabolomes of P. massoniana inoculated with different nematodes. Levels of this compound were measured using UPLC on the 3rd, 7th, and 21st days after inoculation. It was detectable only in the group inoculated with the highly virulent B. xylophilus (nematode strain FCBX), with concentrations of 4.09, 66.77, and 28.8 μg/g, respectively. 7-dehydrocholesterol was undetectable in the sterile water control group (CK), as well as in the groups treated with either highly virulent B. mucronatus (nematode strain BM7) or non-virulent B. mucronatus (nematode strain FCBM).【Conclusion】7-dehydrocholesterol, identified through the metabolomic profiling of P. massoniana, was found to be a specific metabolite following early infection by B. xylophilus. This metabolite's specificity allows it to distinguish B. xylophilus infection from infections caused by other nematodes. Therefore, the detection of 7-dehydrocholesterol levels could serve as an effective tool for the early diagnosis of pine wilt disease.