Hybridization can lead to novel qualitative or quantitative variation of secondary metabolite (SM) expression that can have ecological and evolutionary consequences.•We measured pyrrolizidine alkaloid (PA) expression in the shoots and roots of a family including one Jacobaea vulgaris genotype and one Jacobaea aquatica genotype (parental genotypes), two F1 hybrid genotypes, and 102 F2 hybrid genotypes using liquid chromatography–tandem mass spectrometry (LC-MS/MS).•We detected 37 PAs in the roots and shoots of J. vulgaris, J. aquatica and the hybrids. PA concentrations and compositions differed between genotypes, and between roots and shoots. Three otosenine-like PAs that only occurred in the shoots of parental genotypes were present in the roots of F2 hybrids; PA compositions were sometimes novel in F2 hybrids compared with parental genotypes, and in some cases transgressive PA expression occurred. We also found that PAs from within structural groups covaried both in the roots and in the shoots, and that PA expression was correlated between shoots and roots.•Considerable and novel variation present among F2 hybrids indicates that hybridization has a potential role in the evolution of PA diversity in the genus Jacobaea, and this hybrid system is useful for studying the genetic control of PA expression
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