Abstract: To identify rapidly and accurately the drought resistance of tobacco cultivars, this study used six representative tobacco germplasms to investigate the methods of drought resistance identification and the drought tolerance capacity of different germplasms. A gradient drought stress model was established by controlling the soil relative water content in pot experiments, the response characteristics of six tobacco cultivars to water stress at key growth stages (including physiological phenotypes and survival rates) were evaluated, and drought stress conditions at different growth stages were quantified. Meanwhile, membership function analysis was employed to screen simplified biochemical indicators for drought resistance evaluation. Results showed that identifying drought resistance under the following soil relative water content conditions could accurately reflect the drought resistance of tobacco cultivars: 20% at the seedling stage, 10% at the recovery stage, 20% at the vigorous growth stage, and 15% at the maturation stage. Three drought-resistant cultivars Zhongyan14, Nanjiang3 and K326 were screened out by this method. Higher values of average membership function of proline (Pro) and superoxide dismutase (SOD) were associated with stronger drought resistance in the cultivars. These two indicators can be used as simplified quantitative indices for drought-resistant cultivar evaluation. In conclusion, this study constructed an identification system of tobacco drought resistance, providing a scientific basis and practical method for the rapid identification and evaluation of tobacco drought resistance, thus help accelerate the screening and breeding of drought-resistant tobacco cultivars.