Abstract: Tobacco mosaic virus（TMV） is one of the main diseases in tobacco areas.The TMV-resistance sources used in tobacco production are all from Nicotiana glutinosa, which carries N gene and is immune to TMV,but it is difficult to be widely used in tobacco production due to the poor quality of tobacco leaves due to chain accumulation.Therefore, it is necessary to find new sources of TMV resistance in Nicotiana.In this study, a distant cross was carried out between the flue-cured tobacco variety Honghua dajinyuan and a wild conus tobacco（N. paniculata, PI 555550）,and an interspecific hybrid was obtained.The SSR,GISH and disease resistance identification showed that the hybrid had the specific bands of both parents, and after TMV inoculation, leaf blight reaction was observed.The specific primers of N gene could not be amplified in conus tobacco and hybrid, and the specific primers designed based on Np fragment of N-homologous gene were amplified to 500 bp fragment, but the specific fragment was not amplified in RBST carrying N gene and heart leaf tobacco（N. glutinosa,PI 555507）.The specific fragment was amplified in the hybrid.This study laid a material foundation for obtaining new TMV resistance source.