Drought is one of the numerous environmental factors which seriously affects the growth and development of plants, and it is becoming more severe in many regions of the world due to climate change. To examine physiological responses of tobacco seedling to polyethylene glycol (PEG) stress and provide theoretical basis for evaluating drought resistance and screening tobacco species, the physiological and biochemical indexes of two tobacco cultivars were investigated under the conditions of light drought (LD), intermediate drought (ID) and severe drought (SD). The results show that malondialdehyde (MDA) and hydrogen peroxide (H_2O_2) contents were significantly increased than those of the control, while activities of protective enzymes and chlorophyll contents were obviously increased and then significantly decreased under the ID or SD treatments. The enzyme activities in Yuyan 10 increased more strongly than those in Yuyan 12. Meanwhile, MDA content in Yuyan 10 changed more obviously than that in Yuyan 12, which may be related to the enhanced enzyme activity during droughts, more exactly, a strong negative correlativity between MDA content and enzyme activities was shown under water stress. Subordinate function of drought-resistance and its correlation with drought-resistance indexes of tobacco species were used for a quantitative evaluation of comprehensive drought-resistance. The capabilities of drought-resistance for two different tobacco species were in the order Yuyan 12>Yuyan 10. There was a significant tendency that drought-resistant varieties were more easily adapted to water stress environment than drought-susceptible varieties, and the slight variations of index only occurred in the drought-resistant varieties. The grey correlative degree of drought-resistance with other physiological indexes was in the rank of MDA content, SOD activity, H_2O_2 content, Chl a/Chl b, POD activity, CAT activity and Chl a+b content. Therefore, the SOD activity, MDA content and Chl a/Chl b can be used as effective indicators for the identification of drought resistance, while H_2O_2 content, CAT activity, POD activity and Chl a+b content can be used for reference indicators at seedling stage for tobacco species.