Target spot disease, caused by Rhizoctonia solani (Kühn) (teleomorph Thanatephorus cucumeris (Frank) Donk), significantly reduces tobacco yield and quality. In recent years, the spread of target spot has become a threat to tobacco production in China. However, research on isolation of highly resistant or immune tobacco cultivars to target spot are limited. In this study, 600 tobacco cultivars representing diverse genetic sources from different countries were evaluated for their resistance to tobacco target spot under greenhouse conditions. During screening at a temperature range of 19ºC– 26ºC, two immune cultivars and 11 resistant cultivars were found. The remaining cultivars produced symptoms that designated them as susceptible or highly susceptible at these same temperatures. Since a relatively higher temperature is conducive to R. solani AG-3 infection, which indicates a severe target spot symptom, the response of two immune cultivars and 11 resistant cultivars was further tested at a temperature range of 23ºC – 32ºC. Results indicated that two immune cultivars isolated in a relatively lower temperature range exhibited characteristics that could designate them as resistant, while three resistant cultivars were maintained phenotype among 11 lines tested. Taken together, our analyses tested a large number of tobacco cultivars with different ranges of temperature for response to R. solani AG-3 infection, and resistant lines Reams 51, DF 485, and KY 171 were identified as valuable sources to defend against R. solani AG-3 within a wide range of temperatures.