Post-introduction evolution of plant defense traits is fundamental to several important theories for plant invasiveness. Research on chemical defense traits of invasive plants has focused mostly on targeted chemical analysis, however, such analyses restrict novel insights to known compounds. Here, we provide an untargeted metabolomic analysis of native and invasive Purple Loosestrife populations and we experimentally test if admixture between introduced populations provides a basis for rapid defense chemistry evolution. Invasive populations showed improved growth and generalist herbivore resistance, but lower resistance to a specialist weevil, consistent with the Shifting Defense Hypothesis of plant invasions. Metabolomic profiling revealed large shifts in chemistry between native and invasive populations, including differences in alkaloids and flavonoids. Experimental admixture increased chemical diversity and plant growth in the native populations, indicating its potential to fuel rapid evolution, but admixture did not affect generalist and specialist herbivory. Our untargeted metabolomics analysis provides novel insights in the evolutionary changes in the chemistry of invasive plants, and reveals the chemical associations of a shift from defenses against specialist to generalist herbivores in invasive populations. © 2023, The Author(s), under exclusive licence to Springer Nature Switzerland AG.