Papaver nudicaule L. (Iceland poppy) is widely used for ornamental purposes. A previous study demonstrated the alleviation of lipopolysaccharide-induced inflammation mediated by P. nudicaule extract through nuclear factor-kappa B and signal transducer and activator of transcription 3 inactivation. As isoquinoline alkaloids are chemical markers and bioactive constituents of Papaver species, the present study investigated the alkaloid profile of aerial parts of five P. nudicaule cultivars with different flower colors and a P. rhoeas cropped for two years. A combination of liquid chromatography high-resolution mass spectrometry and molecular networking was used to cluster isoquinoline alkaloids in the species and highlight the possible metabolites. Aside from the 12 compounds, including rotundine, muramine, and allocryptopine, identified from Global Natural Products Social library and reported information, 46 structurally related metabolites were quantitatively investigated. Forty-two and 16 compounds were proposed for chemical profiles of P. nudicaule and P. rhoeas, respectively. Some species-specific metabolites showed similar fragmentation patterns. The alkaloid abundance of P. nudicaule differed depending on the flower color, and the possible chemical markers were proposed. These results show that molecular networking-guided dereplication allows investigation of unidentified metabolites. The derived chemical profile may facilitate evaluation of P. nudicaule quality for pharmacological applications.