Industrial chicory (Cichorium intybusvar.sativum) and witloof (C. intybusvar.foliosum) are crops with an important economic value, mainly cultivated for inulin production and as a leafy vegetable, respectively. Both crops are rich in nutritionally relevant specialized metabolites with beneficial effects for human health. However, their bitter taste, caused by the sesquiterpene lactones (SLs) produced in leaves and taproot, limits wider applications in the food industry. Changing the bitterness would thus create new opportunities with a great economic impact. Known genes encoding enzymes involved in the SL biosynthetic pathway areGERMACRENE A SYNTHASE (GAS), GERMACRENE A OXIDASE (GAO),COSTUNOLIDE SYNTHASE (COS)andKAUNIOLIDE SYNTHASE(KLS). In this study, we integrated genome and transcriptome mining to further unravel SL biosynthesis. We found thatC. intybusSL biosynthesis is controlled by the phytohormone methyl jasmonate (MeJA). Gene family annotation and MeJA inducibility enabled the pinpointing of candidate genes related with the SL biosynthetic pathway. We specifically focused on members of subclade CYP71 of the cytochrome P450 family. We verified the biochemical activity of 14 C. intybusCYP71 enzymes transiently produced inNicotiana benthamianaand identified several functional paralogs for each of theGAO,COSandKLSgenes, pointing to redundancy in and robustness of the SL biosynthetic pathway. Gene functionality was further analyzed using CRISPR/Cas9 genome editing inC. intybus. Metabolite profiling of mutantC. intybuslines demonstrated a successful reduction in SL metabolite production. Together, this study increases our insights into theC. intybusSL biosynthetic pathway and paves the way for the engineering ofC. intybusbitterness.