The green peach aphid, Myzus persicae, is an economically important crop pest that has evolved resistance to numerous insecticides used for control. Recent research has shown that populations of M. persicae in Greece have evolved resistance to flupyradifurone, a butenolide insecticide that acts as a reversible agonist on insect nicotinic acetylcholine receptors (nAChRs). However, the genetic mechanisms underpinning resistance remain unclear. Here we used transcriptomic profiling in combination with in vivo and in vitro functional approaches to show that flupyradifurone resistance in M. persicae is conferred by enhanced expression of the P450 genes CYP6CY3 and CYP6CY4. High levels of flupyradifurone resistance were observed in M. persicae clones from Greece and France, including in a clone that predates the registration of this insecticide. Synergist bioassays with piperonyl butoxide, an inhibitor of P450s, suggested resistance was mediated, in part, by enhanced P450 activity. Subsequent, transcriptome profiling identified several P450s that were overexpressed in the resistant M. persicae clones compared to susceptible clones. Functional characterisation of these P450s revealed that only CYP6CY3 and CYP6CY4 confer resistance in vivo and interact with flupyradifurone in vitro. In two clones with extremely high levels of resistance to flupyradifurone, overexpression of these P450s was accompanied by the R81T mutation in the β1 subunit of the nAChR, known to confer resistance to neonicotinoids and cross-resistance to flupyradifurone. These findings provide new insight into the mechanisms of resistance to flupyradifurone and reveal how generalist P450s can be preadapted to confer cross-resistance to diverse insecticides.