Sesquiterpene lactones in sunflowers, Helianthus spp., are important to interactions with pathogens, weeds, and insects. Across a broad range of Helianthus annuus, differences in composition of sesquiterpene lactones extracted from disc florets were found between wild and cultivated sunflowers and also between distinct groups of inbreds used to produce sunflower hybrids. Discriminant function analysis showed the presence and relative abundance of argophyllone B, niveusin B, and 15-hydroxy-3-dehydrodesoxyfruticin were usually (75%) effective at classifying wild sunflowers, cultivated inbreds, and hybrids. Argophyllone B reduced the larval mass of the sunflower moth, Homeosoma electellum, by >30%, but only at a dose greater than that found in florets. Low doses of mixed extracts from cultivated florets produced a similar (≈40%) reduction in larval mass, suggesting combinations of sesquiterpene lactones act additively. Although the results support a role for sesquiterpene lactones in herbivore defense of cultivated sunflowers, additional information is needed to use these compounds purposefully in breeding.