Bitterness is a key factor that affects the consumption of quinoa products, even if they are nutritious. In this study, a non-targeted metabolomics approach based on UHPLC-Orbitrap-MS was applied to comprehensively profile the characteristic metabolites of twenty-two quinoas. A total of twenty key metabolites were identified correlated with bitterness, among which, fifteen were triterpenoid saponins. In addition, these metabolites bind to the active site of the human bitter taste receptor and are the main compounds that produce the bitter taste of quinoa. Our results contribute to a deeper understanding of the origin of quinoa bitterness and provide directions for optimizing its flavor to improve market acceptance.