Abstract: Pot experiments were conducted at the altitudes of 500 m, 900 m and 1300 m in Enshi Autonomous Prefecture of Hubei to study the differences of photosynthetic characteristics of flue-cured tobacco cultivars with genotypes K326, Yunyan87 and Zhongyan103. The result show that the net photosynthetic rates(Pn) are maximum at full expansion of leaves of the flue-cured tobacco at various altitudes in Enshi, then decrease gradually, which is non-stomatal limitation that causes the decrease of Pn. With the increase of planting altitude, photosynthetic function of leaves is declined more slowly, the photosynthetic function period is prolonged, and the photosynthetic capacity of leaves is raised. Compared with the change of the chlorophyll content, the decrease of activities of RuBPCase is the main factor of the decline of photosynthetic function in tobacco leaves. The chloroplast microstructure is disintegrated gradually after the full expansion of leaves, and with the rising of the altitude, the start time of descending of chloroplast microstructure is delayed. The photosynthetic efficiency of the Zhongyan103 cultivar is highest because of the higher photosynthetic capacity in various elevations.