Abstract: In order to investigate the change of non-targeted metabolites during tobacco maturation process, the upper and middle tobacco leaves from three producing areas (Qujing, Honghe, and Kunming)in Yunnan Province were studied by combining non-targeted metabolomic analysis with gas chromatography-mass spectrometry(GC-MS). The results showed that the relative contents of some sugars, amino acids, and polyphenols in tobacco leaves decreased generally during the aging process, while those of organic acids increased generally. Based on the constitution of the non-targeted metabolites, tobacco maturation could be divided into three stages. The suitable aging periods for the upper and middle leaves of Yunnan tobacco were 21-33 months and 21-27 months respectively. Using partial least squares discriminant analysis(PLS-DA), 16 differential metabolites were screened from early to middle aging stages, which were mainly enriched in the metabolic pathways of carbohydrates. Further 10 differential metabolites were screened from middle to late aging stages, which were mainly enriched in the metabolic pathways of organic acids.