The biosynthesis of the monoterpenes terpinolene and myrcene and the sesquiterpene beta-caryophyllene in roots and leaves of two carrot varieties (Daucus carota L. cultivars Bolero and Kazan) were investigated by in vivo feeding experiments with [5,5-2H2]-mevalonic acid lactone (d2-MVL) and [5,5-2H2]-1-deoxy-D-xylulose (d2-DOX). The volatiles of the tissues were extracted by stir bar sorptive extraction and analyzed using thermal desorption-multidimensional gas chromatography-mass spectrometry. The experiments demonstrate independent de novo-biosynthesis of terpenoids in carrot roots and in carrot leaves. In both plant tissues monoterpenes are biosynthesized exclusively via the 1-deoxy-D-xylulose/2-C-methyl-D-erythritol-4-phosphate (DOXP/MEP) pathway, whereas sesquiterpenes are generated by the classical mevalonic acid pathway as well as by the DOXP/MEP route. A more detailed investigation of carrot root tissues revealed that the biosynthesis of terpenes is mainly localized in the phloem. Nevertheless, in xylem a de novo-biosynthesis of terpenes was detectable as well, even in the absence of oil ducts in this tissue.