Abstract: Tobacco mosaic virus (TMV) and Potato virus Y (PVY) are two destructive plant viruses which cause significant economic losses to tobacco. Breeding resistant cultivars is one of the most important strategies to control these viruses. In this study, the responses to TMV and PVY of 22 tobacco cultivars were evaluated based on resistance index and virus titre obtained from ELISA in both greenhouses and fields during five years (2007-2011). Nicotiana glutinosa resistant to TMV and Virgin A Mutant resistant to PVY were used as resistant checks; NC89 susceptible to both TMV and PVY was used as the susceptible check. The results showed that there were significant differences in disease index and ELISA value among the 22 tobacco cultivars after inoculation. Specifically, 2 cultivars (Coker86 and Jiyan5) were identified to be resistant or moderate resistant to TMV and 3 cultivars (Liaoyan8, Jinxing6007 and Qinyan98) to PVY, while the other cultivars were susceptible or moderate susceptible to TMV or PVY. The resistant cultivars may be used as resistant resources to TMV and PVY in future breeding projects.