Abstract: In this study, tobacco seeds of different varieties were treated with the chemical mutagen, ethyl methane sulfonate (EMS). The germination rate and root lengths of the mutated seeds were measured. The results showed that germination was inhibited at EMS concentrations of 0.1%-1.3%, while the lower concentration EMS could increase the germination rate; the higher concentration EMS could significantly inhibit the germination and root elongation. By the selection criteria of lethal concentration 50, the appropriate concentrations of EMS for treating flue-cured tobacco seeds were 0.35%-0.52% and lethal concentrations for all five varieties were 1.3%. The sensitivity of tested tobacco varieties to EMS in descending order was Yunyan87=Yunyan97 > Honghuada-jinyuan > Zhongyan100 > K326.