Abstract: Low temperature stress frequently occurs at early spring season after transplanting of tobacco seedlings in China, which results in slow growing at seedling stage and early flowering, and seriously leads to fluctuation of yield and quality of tobacco leaf. A new way for identification of cold tolerance in tobacco at seedling stage was present in the paper in order to evaluate the low temperature sensitivity and screen cold tolerant germplasm resources. The screening processes were sequentially done from preparing seedlings with float system, transplanting into pots at three to four true leaves stage, acclimating in room temperature for two to three days at six to eight true leaves stage, stressing the potted plants continuously at 6℃to 8℃for seven days, and then restoring growth at room temperature for ten days. The low temperature susceptible, moderately tolerant and cold tolerant resources were classified based on morphological performance of the resulted tobacco plants at low temperature stress and then restored growing at room temperature. Thirty six accessions including flue-cured tobacco, sun-cured tobacco and wild species were identified with the above method for cold tolerance at seedling stage, four types were easily differentiated according to leaf morphological damage under continuous low temperature for six days, and three types were simply classified based on the restoring growth states during subsequent room temperature condition. Comprehensively, five lines were classified as tolerant to cold, seven lines were classified as moderately tolerant based on the morphological performance of these tobacco resources under low temperature stress and subsequent room temperature condition.