Abstract: In order to establish an efficient and accurate method to identify the resistance of tobacco varieties against tobacco brown spot, four tobacco varieties with different resistance levels were used to study the effects of different concentrations of AT-toxin from Alternaria alternate on tobacco seed germination and seedling root growth after treating for 10, 15 and 20 d. The results showed that the protein and soluble sugar contents in AT-toxin solution were 80.52 μg/mL and 23.39 mg/mL, respectively. With the increase of AT-toxin concentration, the germination rate of tobacco seeds and the percentage of tobacco seedlings with roots' growing into AT-toxin medium (PTS) decreased. Within the AT-toxin concentration and treatment time ranges, the tolerances of tobacco varieties to AT-toxin differed significantly. Comparing with G28, YN116 and Honghuadajinyuan, the PTS of Jingyehuang (resistant variety) were 1.18, 1.75, 6.02 times and 1.02, 1.75, 5.52 times in the 15th and 20th days after 50% AT-toxin treatment, respectively. Through the comprehensive analysis shown, PTS as an indicator of whether tobacco varieties resistant to brown spot is more accurate than seed germination rate, the optimal AT-toxin concentration is 50% and the optimal treatment time is 15-20 d.