Identifying the genetic mechanisms of adaptation requires the elucidation of links between the evolution of DNA sequence, phenotype, and fitness . Convergent evolution can be used as a guide to identify candidate mutations that underlie adaptive traits , and new genome editing technology is facilitating functional validation of these mutations in whole organisms . We combined these approaches to study a classic case of convergence in insects from six orders, including the monarch butterfly (Danaus plexippus), that have independently evolved to colonize plants that produce cardiac glycoside toxins . Many of these insects evolved parallel amino acid substitutions in the α-subunit (ATPα) of the sodium pump (Na /K -ATPase) , the physiological target of cardiac glycosides . Here we describe mutational paths involving three repeatedly changing amino acid sites (111, 119 and 122) in ATPα that are associated with cardiac glycoside specialization . We then performed CRISPR-Cas9 base editing on the native Atpα gene in Drosophila melanogaster flies and retraced the mutational path taken across the monarch lineage . We show in vivo, in vitro and in silico that the path conferred resistance and target-site insensitivity to cardiac glycosides , culminating in triple mutant 'monarch flies' that were as insensitive to cardiac glycosides as monarch butterflies. 'Monarch flies' retained small amounts of cardiac glycosides through metamorphosis, a trait that has been optimized in monarch butterflies to deter predators . The order in which the substitutions evolved was explained by amelioration of antagonistic pleiotropy through epistasis . Our study illuminates how the monarch butterfly evolved resistance to a class of plant toxins, eventually becoming unpalatable, and changing the nature of species interactions within ecological communities .