The greenhouse whitefly Trialeurodes vaporariorum and the tobacco whitefly Bemisia tabaci are global crop pests which cause damage to a wide variety of plants encompassing fruits, vegetables and ornamentals. Their sap-sucking nature combined with their aptitude to act as vector for plant viruses means that control of these organisms is vital for crop management, generally involving the use of synthetic insecticides. Previous work has observed fluctuation in tolerance to insecticides within populations of whiteflies feeding on different host plants. To investigate this, lines of whiteflies were established on five different host plants; pumpkin (Cucurbita pepo), cucumber (Cucumis sativus), tobacco (Nicotiana tabacum), tomato (Solanum lycopersicum) and French bean (Phaseolus vulgaris). Bioassays on these populations revealed large differences in tolerance to insecticides across multiple chemical classes. The general trend observed saw the lines of whiteflies on the nightshade hosts, tobacco and tomato, developing an increased tolerance to pesticides. RNA sequencing from the same whitefly lines revealed large scale changes in gene expression, again particularly in the nightshade-reared lines. Many of the over-expressed genes belonged to detoxification enzyme super-families, which have previously been implicated in insecticide resistance. Sequencing of the first T. vaporariorum genome using 10x genomics discovered multiple novel genes encoding detoxification enzymes. Comparisons between the two whitefly species T. vaporariorum and B. tabaci revealed a 1.5-fold increase in detoxification enzymes present in B. tabaci which could partly explain why this species is considered a greater global pest. Four T. vaporariorum cytochrome-P450s have previously been associated with resistance to insecticides; CYP6CM2, 3 and 4 with neonicotinoids and CYP4G61 with the juvenile hormone analog pyriproxyfen. To identify whether they were able to confer resistance, the three genes were inserted into D. melanogaster lines and bioassayed using the GAL4/UAS system to test their potential to convey resistance to insecticides. No link between the CYP6CM2-4 genes and resistance to neonicotinoids was discovered although an increased tolerance to the natural plant compound nicotine was observed. Similarly, CYP4G61 did not appear to confer resistance to pyriproxyfen in flies, a result confirmed by further microsomal analysis.