Abstract: To clarify the roles of non-volatile organic acids and higher fatty acids in the quality evaluation of upper leaves of flue-cured tobacco, 111 samples of grade B2F from 27 tobacco planting regions in 12 provinces of China were measured by gas chromatography-mass spectrometry. The differences in the contents of non-volatile organic acids and higher fatty acids in the leaf samples and their relationships with the routine chemical indexes as well as sensory quality were analyzed with multivariate statistical methods. The near-infrared rapid measurement models were established by the integrating-sphere diffuse-reflection technique. The results showed that: 1) Both the contents and distribution patterns of the measured non-volatile organic acids and higher fatty acids in the tested upper flue-cured leaf samples were different. 2) Palmitic acid content was closely correlated to the routine chemical indexes, and there was a high correlation between palmitic acid content and the contents of total sugar and reducing sugar, with correlation coefficients of 0.650 7 and 0.641 4 respectively. 3)Palmitic acid had significant effects on 10 sensory evaluation indexes, malic acid mainly affected the smoothness, clarity, aroma quantity, offensive odor and total sensory score, and fumaric acid mainly affected smoothness, clarity, aftertaste and total sensory score. The sensory scores of tobacco leaves with palmitic acid content higher than 3.00 mg/g, malic acid content less than or equal to 65.00 mg/g, and fumaric acid content higher than 0.07 mg/g were significantly higher. 4) The correlation coefficients of the near-infrared-corrected and cross-validated models for palmitic acid, malic acid, and fumaric acid were above 89%. These models could be used to rapidly detect the palmitic acid, malic acid, and fumaric acid contents in upper flue-cured leaves. In conclusion, palmitic acid, malic acid, and fumaric acid could be used as important quality evaluation indexes for upper flue-cured tobacco leaves.