Abstract: An integrated strategy that combining untargeted metabolomics based on ultrahigh performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS), targeted amino acid analysis and chemometrics was developed to achieve the metabolic profiling analysis of different parts (pileus and stipe) of Pholiota adiposa fruiting body. The HRMS data of Pholiota adiposa were acquired in positive ion mode and directly analyze by AntDAS-DDA, a recently developed automatic data analysis software in chemometrics. In addition, the contents of 16 free amino acids in the samples were quantified according to the national standard methods. The metabolomics characteristics across different parts of the Pholiota adiposa fruiting body were investigated using heatmap analysis, radar chart analysis, hierarchical cluster analysis (HCA) and principal component analysis (PCA). The results indicated that a total of 73 involatile differential metabolites were identified, encompassing amino acids, nucleotides, purines, alkaloids, terpenoids, lipids, etc., 12 of those compounds were further verified by standards. Except for tryptophan, which was more abundant in the stipe, contents of lysine, histidine, arginine, tyrosine, phenylalanine, vitamin B5, and vitamin B2 were consistently slightly higher in the pileus than in stipe. Nucleotides such as adenosine and guanosine, along with purines like guanine and hypoxanthine, also showed higher content in the pileus. Umami amino acids were the most abundant category across all parts of the Pholiota adiposa fruiting body, constituting over 30% of the total free amino acids. Notably, the content of umami amino acids in the pileus was significantly higher than in the stipe. the pileus, stipe and whole fruiting body of Pholiota adiposa could each be clearly clustered and distinguished. This research provides a scientific basis for the identification of nutritional characteristics and resource utilization of different parts of Pholiota adiposa, while offering a novel analytical strategy for studying chemical components in other edible fungi.