Abstract: Tobacco black shank is one of the most destructive diseases in tobacco. It is highly significance to establish an identification method on resistant of tobacco black shank using inoculation in a float seeding system. Pathogenicity determination was conducted and No 162 isolate was screened to study resistant identification of tobacco black shank disease. A resistant identification method in float seeding system was studied and the influence was analysized for different inoculation condition on identification results. The results showed that 40 d seedling inoculation, water temperature 28 ℃ and wound inoculation was fit to resistant identification and evaluation of tobacco varieties. Three inoculation methods (float system, pot experiment, and field nursery) were compared with regard to resistant identification. An identification method on resistant of tobacco black shank using inoculation in a float seeding system was established.