【Objective】Comprehensive evaluation on drought resistance of different flue-cured tobacco varieties at seedling stage was performed with subordinate function values analysis， in order to provide theoretical references for breeding and cultivation of tobacco with drought-resistance. 【Method】25 main varieties in Yunnan Province and China were taken as materials. Floating seedlings of tobacco were exposed to drought stress with controlled water supply， followed by measurement of survival rates. In addition， changes of physiological and biochemical characteristics during stress treatment （3， 6， and 9 d） were identified， including relative water content， relative conductivity and content of MDA. Then final comprehensive evaluation on drought tolerance of different varieties was analyzed with subordinate function， using average membership function values of each index. 【Result】The results indicated that the survival rates of seedlings and leaf water content significantly reduced under drought stress， whereas relative electric conductivity， contents of proline， soluble sugar， as well as MDA increased in tobacco leaves. Moreover， changes in indexes showed obvious differences in the tested varieties. Finally， among all the selected 25 tobacco varieties， K326 exhibited the highest drought resistance， due to its highest average membership function values （0.755）， and next ones were RG17 （0.668） and Panyuanhuang （0.650）. In contrast， Yunyan 97 and Yunyan 202 showed less drought tolerance， with their average membership function values below 0.400. 【Conclusion】K326， RG17 and Panyuanhuang were identified as drought-tolerant varieties at seedling stage， which are suitable for extensive planting.