Specialized metabolism plays a crucial role in plant interactions with biotic and abiotic environments, and its characterization in crops is an essential objective for optimizing ecosystem services in breeding programs. However, our knowledge of specialized metabolism in cultivated Brassica species remains insufficient to fully support these breeding strategies. In this study, we conducted a detailed exploration of both constitutive and stress-induced specialized metabolites in response to CuCl₂ in the leaves and roots of 20 Brassica rapa and B. oleracea accessions using UHPLC-HRMS/MS-based untargeted metabolomics. Our analysis revealed a wide range of species-specific chemical signatures and allowed the putative annotation of nearly 200 compounds belonging to various chemical families, including flavonoids, glucosinolates, phenolamides, indoles, amino acids, oxylipins and coumarins. A detailed interpretation of ion fragmentation patterns uncovered both inter- and intra-specific structural variations within these chemical families, providing deeper insights into the phytochemical diversity of B. rapa and B. oleracea species. This study also enabled the identification of previously unreported compounds in these species, including the carotenoid-derivatives blumenols, flavonols decorated with rhamnoside moieties or pentahexosylated flavonols. Finally, we showed that these resources, combined with the high-quality genetic data available for these accessions, allowed the identification of genetic variants of MAM2 genes possibly involved in the control of 4C or 3C/4C-aliphatic glucosinolate chemotypes among B. oleracea accessions. This work thus lays the foundation for future studies on functional genomics of specialised metabolism in Brassica species in a chemical ecology perspective, and aiming to harness metabolic diversity in breeding programs.