Abstract: Granville wilt caused by Ralstonia solanacearum is a major limiting factor in tobacco production in China. The most economically feasible strategy to control the disease is the use of resistant cultivars. In this study, the potential of inoculation of in vitro plantlet as a rapid and reliable method for screening tobacco cultivar against R. solanacearum was investigated. In vitro plantlets of six tobacco cultivars with diverse resistance levels were inoculated with 3×10⁶ cfu/mL cell suspension of R. solanacearum strain Tb2K9-11. Twenty-one days after inoculation, the disease index of cultivar Honghuadajiyuan, Cuibi No.1, Yunyan 85, K326, G80 and Yanyan 97 was 100.0, 90.5, 88.8, 86.6, 56.0 and 53.8, respectively. There were significant correlations between in vitro plantlet assay and field trial (r=0.936). The result showed that the in vitro screening assay can help in improving tobacco breeding efficiency.