Deploying resistant cultivars is an economical and essential management method in controlling viral diseases, and there are several mutational resources for tobacco. In the present study, the inoculation of tobacco plants with tobacco viruses was performed in a greenhouse from 2011 to 2014 to identify mutants resistant to tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV). The high-throughput screening included seeding uniformly, transplanting in seedbeds, inoculating by cloth brushes and reporting symptoms based on disease indices. A total of 4000 second generation segregating (M2) mutants of tobacco cultivar Zhongyan100 were screened. Seeds from highly resistant mutant M2 plants were selected and planted separately. The M3 were grown and mutational stability was measured. For TMV, ten highly resistant plants were selected in the M2 generation and the mutation rate was 0·012%. In the M3 generation, there were seven mutants with hereditary high resistance and, according to the results of real-time polymerase chain reaction, the N gene was detected in all seven M3. Two hereditary immune M4 mutants, one of which was a male sterile line, were identified and evaluated in the glasshouse and in the field. For CMV, seven highly resistant plants were selected from the M2 generation and the mutation rate was 0·009%. In the M3 generation, there was one mutant with hereditary high resistance. The results indicate that hereditary mutants may be identified in the M4 generation and back-crossed to wild-type Zhongyan100 to identify anti-viral genes.