Cassava (Manihot esculenta Crantz) is an important starch-rich crop, but the storage roots only have a short shelf-life due to post-harvest physiological deterioration (PPD) which includes the over-production and polymerisation of hydroxycoumarins. Key aspects of coumarin secondary metabolite biosynthesis remain unresolved. Here we exploit this accumulation of hydroxycoumarins to test alternative pathways for their biosynthesis. Using isotopically labelled intermediates (p-coumarate-2-13C, caffeate-2-13C, ferulate-2-13C, umbelliferone-2-18O and esculetin-2-18O), we show that the major biosynthetic pathway to scopoletin and its glucoside scopolin in cassava roots during PPD is through p-coumaric, caffeic and then ferulic acids. An alternate pathway through 2',4'-dihydroxycinnamate and umbelliferone leads to esculetin and esculin. We have used C18O2-carboxylate labelled cinnamic and ferulic acids, and feeding experiments in an atmosphere of 18O2, to investigate the o-hydroxylation and cyclisation steps. We demonstrate that the major pathway is through o-hydroxylation and not via a proposed spirolactone-dienone intermediate.