Abstract: In order to clarify the differences of major quality characters among flue-cured tobacco leaves grown in different ecological regions of Yunnan province, appearance quality, chemical composition and sensory quality among 482 samples of middle flue-cured tobacco leaves taken from east, center, west and southwest regions of Yunnan were systematically analyzed and comparatively studied. The correlation between smoking quality and chemical composition was further carried out. Results showed that no significant differences were found in the nicotine content, sugar-alkali ratio, K/Cl and the comprehensive appearance quality among middle leaves from four different regions, and that middle leaves from east, west and southwest Yunnan were comparable in overall smoke quality. The fresh-sweet and green notes all decreased gradually in leaves from center, east, west, and southwest of Yunnan, while the cigarette impact increased gradually in leaves from the east, the center and the southwest of Yunnan. The scores of sweet fragrance, aroma quality, aroma quantity, aftertaste and the comprehensive scores of smoke quality were given higher for leaves from center Yunnan than for the others. The woody note of leaves in southwest Yunnan was very significantly higher than those from eastern Yunnan and its potassium content together with smoke strength were higher than those in western Yunnan. The total sugar content in leaves from west Yunnan was significantly higher than those from center Yunnan, and the reducing sugar as well as potassium content were significantly higher than leaves from east and center of Yunnan. However, its total nitrogen content was significantly lower than samples from other regions. Stepwise regression analysis showed that comprehensive smoking quality of tobacco leaves in the east and west regions of Yunnan could be improved by reducing sugar-alkali ratio and increasing nitrogen-alkali ratio.