Tobacco bushy top disease (TBTD) is one of the serious diseases affecting tobacco (Nicotiana tabacum L.) production in African countries such as Malawi, Zambia, Tanzania and Ethiopia. This disease is caused by a mixed infection of tobacco bushy top virus (TBTV, genus Umbravirus) and its helper virus for aphid transmission (a member of the genus Polerovirus). In a recent study, we demonstrated that loss-of-function of eukaryotic translation initiation factor eIF(iso)4E in tobacco conferred resistance to TBTV by sap inoculation in a greenhouse. The objective of this study was to evaluate the loss-of-function tobacco lines of eIF(iso)4E for resistance to TBTD by aphid transmission under a natural environment and for agronomic traits in fields. Field tests for resistance to TBTD were conducted for three seasons in fields of the Agricultural Research and Extension Trust in Malawi. Agronomic traits were examined at fields of the JT Leaf Tobacco Research Center in Japan. The loss-of-function tobacco lines of eIF(iso)4E of both flue-cured and Burley types were used for this study. Wild type lines for eIF(iso)4E were used as a control. Regarding the incidence of TBTD, the loss-of-function lines of eIF(iso)4E were significantly lower than the control lines in almost all field tests. Regarding plant height and total leaf numbers at the flowering period and yield of cured-leaves, the loss-of-function lines of eIF(iso)4E were not significantly different from the control lines. These results suggest that loss-of-function of eIF(iso)4E in tobacco confers resistance to TBTD under a natural environment and that it does not adversely affect agronomic traits. Future cultivation of tobacco varieties with loss-of-function of eIF(iso)4E will contribute to stable leaf tobacco production in regions adversely affected by economic losses from TBTD.