Tobacco bushy top disease (TBTD) is a viral disease of tobacco (Nicotiana tabacum L.) caused by mixed infection of Tobacco bushy top virus or Ethiopian tobacco bushy top virus and a helper virus. Despite its damage to tobacco, practical genetic resources for disease resistance have not been found. Here, we report that a mutation of tobacco eIF(iso)4E genes (eIF(iso)4E-S and eIF(iso)4E-T), which encode eukaryotic translation initiation factors, confers resistance (reduced susceptibility) to TBTD caused by a virus from Malawi (designated as tobacco bushy top virus Malawi isolate, TBTV-MW). RNAi lines in which eIF(iso)4E genes were silenced showed reduced susceptibility to TBTV-MW. We also tested chemically-induced single (eIF(iso)4E-S or eIF(iso)4E-T) and double (eIF(iso)4E-S and eIF(iso)4E-T) nonsense mutants for resistance to TBTV-MW. Suppression of eIF(iso)4E-S showed reduced susceptibility, and the resistance of the double mutant tended to be even stronger. eIF(iso)4E mutants also showed reduced susceptibility to TBTV-MW transmitted by aphids. To the best of our knowledge, the eIF(iso)4E-S mutant is the first genetic resource for TBTD resistance breeding in tobacco. Copyright © 2020 by JAPANESE SOCIETY OF BREEDING.