The physiological and ultrastructural changes of tobacco varieties with different chilling tolerances under chilling stress were investigated in this study. Twenty tobacco varieties were separated into three groups; chilling tolerant, intermediate chilling tolerant, and chilling sensitive varieties, with eight cluster analysis methods based on seven morphological parameters measured at low temperature. After a chilling stress for five days at 11°C, the activities of ascorbate peroxidase (APX), peroxidase (POD), superoxide dismutase (SOD), the concentrations of malondialdehyde (MDA), hydrogen peroxide (H2O2), superoxide anion radical (O2-), chlorophyll ‘a’ and ‘b’, total soluble sugar, and soluble protein were measured in the three varieties with different chilling tolerance. The cell ultrastructure of the three varieties was observed by transmission electron microscope. The results indicated that the values of APX, POD, SOD, MDA, H2O2, O2-, total soluble sugar, and soluble protein were increased under chilling stress, while the values of chlorophylls a and b were decreased in all the three varieties. The values of APX, POD, SOD, total soluble sugar, and protein in chilling tolerant variety were higher than chilling sensitive variety, the values of MDA, H2O2, O2-, and chlorophyll a and b in chilling tolerant variety were lower than those in chilling sensitive variety at 11°C. The separation of the cell membranes and chloroplasts from the cell wall along with an increase in the number of lipid droplets as well as the depletion of starch granules was observed in all tobacco varieties under the chilling stress, and these ultrastructural changes in chilling sensitive variety were more visible than chilling tolerant and intermediate chilling tolerant varieties. Key words: Tobacco, chilling stress, physiological changes, ultrastructure.