Hybridization between plant species can have a number of biological consequences; interspecific hybridization has been tied to speciation events, biological invasions, and diversification at the level of genes, metabolites, and phenotypes. This study aims to provide evidence of transgressive segregation in the expression of primary and secondary metabolites in hybrids between Jacobaeavulgaris and J. aquaticus using an NMR-based metabolomic profiling approach. A number of F2 hybrid genotypes exhibited metabolomic profiles that were outside the range encompassed by parental species. Expression of a number of primary and secondary metabolites, including jacaronone analogues, chlorogenic acid, sucrose, glucose, malic acid, and two amino acids was extreme in some F2 hybrid genotypes compared to parental genotypes, and citric acid was expressed in highest concentrations in J. vulgaris. Metabolomic profiling based on NMR is a useful tool for quantifying genetically controlled differences between major primary and secondary metabolites among plant genotypes. Interspecific plant hybrids in general, and specifically hybrids between J. vulgaris and J. aquatica, will be useful for disentangling the ecological role of suites of primary and secondary metabolites in plants, because interspecific hybridization generates extreme metabolomic diversity compared to that normally observed between parental genotypes.