Segregating plant hybrids often have more ecological and molecular variability compared to parental species, and are therefore useful for studying relationships between different traits, and the adaptive significance of trait variation. Hybrid systems have been used to study the relationship between the expression of plant defense compounds and herbivore susceptibility. We conducted a western flower thrips (WFT) bioassay using a hybrid family and investigated the relationship between WFT resistance and pyrrolizidine alkaloid (PA) variation. The hybrid family consisted of two parental (Jacobaea vulgaris and Jacobaea aquatica) genotypes, two F 1 genotypes, and 94 F 2 hybrid lines. The J. aquatica genotype was more susceptible to thrips attack than the J. vulgaris genotype, the two F 1 hybrids were as susceptible as J. aquatica, and susceptibility to WFT differed among F 2 hybrid lines: 69 F 2 lines were equally susceptible compared to J. aquatica, 10 F 2 lines were more susceptible than J. aquatica and 15 F 2 lines were as resistant as J. vulgaris or were intermediate to the two parental genotypes. Among 37 individual PAs that were derived from four structural groups (senecionine-, jacobine-, erucifoline-and otosenine-like PAs), the Noxides of jacobine, jaconine, and jacoline were negatively correlated with feeding damage caused by WFT, and the tertiary amines of jacobine, jaconine, jacoline, and other PAs did not relate to feeding damage. Total PA concentration was negatively correlated with feeding damage. Among the four PA groups, only the total concentration of the jacobine-like PAs was negatively correlated with feeding damage. Multiple regression tests suggested that jacobine-like PAs play a greater role in WFT resistance than PAs from other structural groups. We found no evidence for synergistic effects of different PAs on WFT resistance. The relationship between PA variation and WFT feeding damage in the Jacobaea hybrids suggests a role for PAs in resistance to generalist insects.