The cyanogenic glucoside profile of Eucalyptus camphora was investigated in the course of plant ontogeny. In addition to amygdalin, three phenylalanine-derived cyanogenic diglucosides characterized by unique linkage positions between the two glucose moieties were identified in E. camphora tissues. This is the first time that multiple cyanogenic diglucosides have been shown to co-occur in any plant species. Two of these cyanogenic glucosides have not previously been reported and are named eucalyptosin B and eucalyptosin C. Quantitative and qualitative differences in total cyanogenic glucoside content were observed across different stages of whole plant and tissue ontogeny, as well as within different tissue types. Seedlings of E. camphora produce only the cyanogenic monoglucoside prunasin, and genetically based variation was observed in the age at which seedlings initiate prunasin biosynthesis. Once initiated, total cyanogenic glucoside concentration increased throughout plant ontogeny with cyanogenic diglucoside production initiated in saplings and reaching a maximum in flower buds of adult trees. The role of multiple cyanogenic glucosides in E. camphora is unknown, but may include enhanced plant defense and/or a primary role in nitrogen storage and transport.Graphical abstractA total of six cyanogenic glucosides occur in Eucalyptus camphora tissues including the diglucosides eucalyptosin B and eucalyptosin C.Highlights► Six cyanogenic glucosides occur in Eucalyptus camphora, including four diglucosides. ► Two cyanogenic diglucosides are described, named eucalyptosin B and eucalyptosin C. ► Relative abundance of cyanogenic glucosides varies with both tissue type and ontogeny. ► Polymorphism observed in timing of initiation of prunasin biosynthesis in seedlings.