The objectives of this study were to ascertain the impact of endogenous production of trichome-exuded acylsugars on insects and insect transmitted virus by evaluating tomato lines and their hybrids bred for acylsugar production under field settings on whiteflies and the whitefly-transmitted tomato yellow leaf curl virus. Specifically, we utilized a diverse array of tomato lines and hybrids bred for changes in acylsugar amount or type, grown in three field trials under natural whitefly and virus pressure, to investigate whether the amount of accumulated acylsugars and or the chemical profile of the acylsugars were associated with greater resistance to whiteflies and reduced incidence of tomato yellow leaf curl virus. There was considerable variation in the abundance of whitefly eggs and nymphs and incidence of tomato yellow leaf curl virus across experiments and between entries. Increasing amount of acylsugars accumulated by the tomato entries was associated with a reduction in the abundance of whitefly eggs and nymphs and a reduction in the incidence of tomato yellow leaf curl virus. Additionally, we identified lines with changes in several acylsugar fatty acids that were associated with decreased abundance of whitefly eggs and nymphs and reduced incidence of tomato yellow leaf curl virus. These results inform the utility of acylsugars as a host plant defense system for improving resistance to whiteflies and their transmitted viruses, with potential for reducing insecticides as a control method for whiteflies and provide breeding targets for optimization of existing acylsugar tomato lines to create lines with the most efficacious amount and chemistry of acylsugars.