Abstract: In order to find out an approach to rapidly identify resistant tobacco germplasm to Alternaria alternata, tobacco varieties Jingyehuang, Beinhart1000-1, NC89 and G140 were used as test materials. Using toxins produced by brown spot fungus (Alternaria alternata), we carried out a comparative test to choose a better screening method. Three kinds of screening methods were designed: toxins liquid soaking root of tobacco, toxins liquid soaking seeds of tobacco, and toxin-containing medium screening method. To verify the reliability of the toxin-containing medium screening method, Correlation between laboratory test results and field data on tobacco varieties Jingyehuang, Beinhart1000-1, NC89, G140, K326, G28 and Zhongyan100 was analyzed, and correlation coefficient of -0.9588 was found, which suggests the effectiveness of laboratory method is close to those of field.