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Spodoptera frugiperda, the fall armyworm (FAW), is an important polyphagous agricultural pest feeding on nearly 350 host plants. FAW is undergoing incipient speciation with two well-characterized host-adapted strains, the "corn" (CS) and "rice" (RS) strains, which are morphologically identical but carry several genes under positive selection for host adaptation. We used non-targeted metabolomics based on gas chromatography/mass spectrometry to identify differences in metabolite profiles of the larval gut of CS and RS feeding on different host plants. Larvae were fed on artificial diet, maize, rice, or cotton leaves from eclosion to the sixth instar, when they had their midgut dissected for the analysis. This study revealed that the midgut metabolome of FAW varied due to larval diet and differed between the FAW host-adapted strains. Additionally, we identified several candidate metabolites that may be involved in the adaptation of CS and RS to their host plants. Our findings provide clues toward the gut metabolic activities of the FAW strains.