Drought is one of the many environmental factors which affect the growth and development of plants in many regions of the world due to climate change. The effect of moderate drought stress on seedling biomass, root morphology, stomatal characteristics, chloroplast ultra-structure and photosynthetic parameters of four flue-cured tobacco cultivars (Y6, Y10, Y12 and ZY100) were investigated in a hydroponic experiment with polyethylene glycol (PEG-6000) simulating moderate drought stress to evaluate drought tolerance of different flue-cured tobacco varieties. The results showed that the roots and leaves of different cultivars of tobacoo under moderate drought stress had different physiological responses. (1) A close relationship was noted between root growth and water supply in this study. The moderate drought stress stimulated root growth of flue-cured tobacco seedling, while it inhibited shoot growth. Root to shoot ratio of the 4 flue-cured tobacco cultivars significantly increased under drought stress conditions compared with that of control. Root biomass, total root length, root surface area and root volume of Y6 and Y12 significantly increased while root average diameter decreased. Then roots of Y10 and ZY100 were sensitive to moderate drought stress where root morphology index increased little or even decreased. Although the average diameter of the 4 flue-cured cultivars seedlings decreased significantly, root surface area of Y10 increased significantly. (2) Drought stress significantly decreased stomatal density while increasing both stomatal length and width of Y6, Y12 and Y10. There was a significant difference in stomatal density of ZY100 seedlings between the control group and the drought stress group. However, no significant difference was observed in stomatal width of the four flue-cured tobacco cultivars seedlings. Total pore area per leaf area of ZY100 significantly increased under drought stress. (3) Chloroplast ultra-structure of Y6 and Y12 was slightly changed under drought stress and the change was more obvious in Y10 and ZY100 cultivars. The shape of chloroplast in mesophyll cells changed and separated from cell wall, which destroyed chloroplast integrity. The average length, length-width ratio and area index of chloroplast decreased significantly for ZY100 seedlings under drought stress. (4) Net photosynthetic rate (P_n), transpiration rate (T_r) and stomatal conductance (G_s) of the 4 cultivars seedlings declined, while intercellular CO_2 concentration (Ci) increased significantly in response to drought stress. This indicated that the decline in photosynthesis was as a result of the absence of stomatal restriction under moderate drought stress. The differences between drought group and control group were significant for Y10 and ZY100 seedlings. (5) Under drought stress, the chlorophyll contents of Y6 and Y12 increased significantly, while that of Y10 and Z100 increased slightly or even decreased. The comprehensive analysis showed that Y6 and Y12 were drought resistant cultivars, and Y10 and ZY100 were drought sensitive cultivars. Drought resistance ability of the 4 flue-cured tobacco cultivars was in the order of Y6 > Y12 > Y10 > ZY100 at seedling stage.