Abstract: High-throughput resistance evaluation in vitro is important for the screening and breeding of tobacco varieties resistant to tobacco brown spot disease. In this study, detached leaves at the seedling stage of 15 tobacco germplasm materials with different resistance to tobacco brown spot disease were inoculated with plugs of Alternaria alternata. The lesion area was used to evaluate the disease resistance of the 15 tobacco germplasm materials after inoculation, and the tobacco resistance identified at the seedling stage was compared with that at the adult stage in the field. The results showed that the lesion areas of 15 tobacco varieties were significantly different after inoculation, and the lesion areas of the susceptible varieties was obviously larger than that of the resistant varieties. The resistance identified at the seedling stage and at the adult stage was similar. At the same location with three years' field experiments, the correlation coefficients between lesion areas and disease indexes were 0.78, 0.74 and 0.66, all at a significant level. In conclusion, this method of inoculation of fungal plugs on detached seedling leaves can be used to effectively evaluate the resistance of tobacco varieties to brown spot disease at early stages, and lesion area can be taken as an index to accurately indicate the resistance of different tobacco varieties to brown spot disease.