Abstract: To investigate the characteristic flavors of Tricholoma matsutake of different size grades and tissues, volatile organic compounds (VOCs) and non-volatile taste compounds (NVTCs) were analyzed using electronic nose, gas chromatography-ion mobility spectrometry (GC-IMS), amino acid analysis, and high-performance liquid chromatography. The results showed that pileus and stipe tissues could be rapidly distinguished by electronic nose. A total of 75 VOCs were identified by GC-IMS, including alcohols, ketones, aldehydes, esters, hydrocarbons, and furans, with the stipe tissues exhibiting significantly higher total VOC content than the pileus. The orthogonal partial least squares discriminant analysis (OPLS-DA) model revealed that the different size grades and tissues exhibited distinct VOC concentration differences. Based on the variable importance in projection values from the OPLS-DA model and the relative odor activity value, six key aroma markers were identified: 1-octen-3-ol, 1-octen-3-one, (E)-2-octenal, hexanal, 2-ethylbutanal, and 3-methylbutanal. With an increase in fruiting body size, the VOCs in the pileus of T. matsutake transformed from a simple mushroom-like aroma to a complex aroma profile, whereas those in the stipes remained relatively stable. Analysis of the non-volatile flavor compounds showed that glutamic acid was the main umami-contributing component of T. matsutake. The equivalent umami concentration（EUC） of the pileus was significantly higher than that of the stipe tissues, and the highest EUC was found in the pileus of the 3-5 cm T. matsutake. These results provide a scientific reference for establishing flavor-oriented grading standards for T. matsutake.