Abstract: The changes in leaf photosynthetic characteristics of two different tolerant tobacco cultivars at seedling stage under low temperature stress and subsequent room temperature condition were investigated in the paper. The results showed that the seedling growth of the moderate tolerant tobacco cultivar K326 and the sensitive one Yunyan 97 was nearly inhibited completely at low temperature stress from 4℃ to 6℃, while the growth rate of K326 was significantly higher than that of Yunyan 97 under subsequent room temperature. The leaf photosynthesis at seedling was reduced under low temperature stress, and the inhibitory effects became worse with the elongation of stress treatment. The leaf photosynthetic indexes such as net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci), transpiration rate (Tr), water use efficiency (WUE), stomatal limitation (Ls), maximum photochemical efficiency of photosystem II (Fv/Fm), quantum yield of electron transport (ΦPSII), photochemical quenching co-efficient (qP), apparent electron transfer rate (ETR) and non-photochemical quenching co-efficient (NPQ) under low temperature for 6 days were significantly different with the untreated control. The leaf photosynthesis of tobacco seedlings was raised under subsequent room temperature condition, and the increasing range in K326 was much more than that of Yunyan 97. Although the leaf photosynthesis of the both two cultivars under subsequent room temperature for 9 days was not restored to the levels before low temperature stress, the leaf photosynthetic indexes in the moderate tolerant cultivar K326 were only a little lower than untreated seedlings, while these indexes in the sensitive cultivar Yunyan 97 were still significantly different with the untreated ones.