show Abstracthide AbstractPhytochemical diversity is shaped by coevolutionary cycles as specialization in herbivores imposes diversifying selection on plant chemical defenses. Plants in the speciose genus Erysimum (Brassicaceae) produce both ancestral glucosinolates and evolutionarily novel cardenolides as potent defenses. Here we test macroevolutionary hypotheses about the interplay of these potentially redundant defenses across the genus. We sequenced and assembled the genome of E. cheiranthoides and foliar transcriptomes of 47 additional Erysimum species to construct a highly resolved phylogeny, which showed that cardenolide diversity arose rapidly rather than gradually. Erysimum leaves contained both glucosinolates and cardenolides, with their concentrations, inducibility, and compound diversity varying independently. Furthermore, closely related species shared similar chemotypes for cardenolides, but not glucosinolates, suggesting that selection on these defenses acts at different scales. Ancestral and novel defenses in Erysimum thus appear to have evolved independently in response to different environments, and likely contributed to the evolutionary success of this widespread genus.