Chilling injury during tobacco seedling stage is an important problem affecting tobacco agricultural production, and enhancement of the chilling resistance by various agronomic measures has practical significance. In this study, using two main cultivars Yunyan97 and K326 as the experimental materials, the seedlings were pretreated with salicylic acid (SA) at concentrations 0, 0.5, 1.0 and 1.5 mmol/L. Then the pretreated seedlings were transferred to 10℃ for 12 days for chilling treatment. Compared with the control without SA pretreatment, the SA pretreatment could significantly reduce the malondialdehyde (MDA) content and ratio of electrolyte leakage, slowed down the consumed rate of dry matter, increased chlorophyll content and root vigor, improve root length, surface area, volume and number of roots, promoted the growth of root system under the chilling stress, and finally enhanced the chilling resistance of 'Yunyan 97' and 'K326' seedlings. The optimum SA concentration of different tobacco varieties varied, that for 'Yunyan 97' and 'K326' was 1.5 mmol/L and 1.0 mmol/L respectively. After the SA pretreatment, the content of reduced ascorbic acid (ASA) and glutathione (GSH) in the antioxidant defense system in the seedlings remained a higher level, but oxidized ASA and GSH maintained a lower level, and ratio of the reduced antioxidants in total antioxidants increased. Ascorbate peroxidase (APX), catalase (CAT), guaiacol peroxidase (GPX), glutathione reductase (GR) and superoxide dismutase (SOD) maintained high activities compared with the control before and during the chilling treatment. The results indicated that after the SA pretreatment, the seedlings could maintain higher reduced antioxidant level and antioxidant enzyme activity, and remain higher antioxidant capacity, which would lay an important biochemical foundation for SA-induced chilling resistance in tobacco seedlings. The results would not only have reference significance for raising chilling-resistant tobacco seedlings, but also referential value for culture of seedlings and early cultivation of other crops and vegetables.