The presented results show the usefulness of Raman spectroscopy in the investigation of polyacetylenes in carrot root. The components are measured directly in the plant tissue without any preliminary sample preparation. Compared with the strong polyacetylene signals the spectral impact of the surrounding biological matrix is weak, except for carotenoids, and therefore it does not contribute significantly to the obtained results. Three different Raman mapping techniques applied here have revealed essential information about the investigated compounds. Using point acquisition several spectra have been measured to demonstrate the complex composition of the polyacetylene fraction in carrot root. The molecular structures of falcarinol, falcarindiol and falcarindiol 3-acetate are similar but their Raman spectra exhibit differences demonstrated by the shift of their –CC– mode. Line mapping performed along the diameter of transversely cut carrot roots has been used to investigate the relative concentration of polyacetylenes and carotenoids. An area map provides detailed information regarding the distribution of both components. It has been found that high accumulation of polyacetylenes is located in the outer section of the root, namely the pericyclic parenchyma, and in the phloem part close to the secondary cambium. The highest concentration of carotenes is seen in the immediate vicinity to polyacetylene conglomerates.