This paper presents an approach to simultaneously analyze polyacetylenes, carotenoids, and polysaccharides in carrot (Daucus carota L.) roots by means of Raman spectroscopy. The components were measured in situ in the plant tissue without any preliminary sample preparation. The analysis is based on the intensive and characteristic key bands observed in the Raman spectrum of carrot root. The molecular structures of the main carrot polyacetylenes, falcarinol and falcarindiol, are similar, but their Raman spectra exhibit specific differences demonstrated by the shift of their -C[triple bond]C- mode from 2258 to 2252 cm(-)(1), respectively. Carotenoids can be identified by -C=C- stretching vibrations (about 1520 and 1155 cm(-)(1)) of the conjugated system of their polyene chain, whereas the characteristic Raman band at 478 cm(-)(1) indicates the skeletal vibration mode of starch molecule. The other polysaccharide, pectin, can be identified by the characteristic band at 854 cm(-)(1), which is due to the -C-O-C- skeletal mode of alpha-anomer carbohydrates. The Raman mapping technique applied here has revealed detailed information regarding the relative distribution of polyacetylenes, carotenoids, starch, and pectin in the investigated plant tissues. The distribution of these components varies among various carrot cultivars, and especially a significant difference can be seen between cultivated carrot and the wild relative D. carota ssp. maritimus.