Hand-dissection of mature grains from three common wheat cultivars (Triticum aestivum L.: Caphorn, Crousty, and Recital) and one durum wheat (T durum Desf., Ardente) was performed to obtain pure samples of aleurone layer. hyaline layer, outer pericarp, and a composite layer made up of testa+inner pericarp. Vibrational spectral signatures were collected on both sides of the layers by ATR-FTIR and Raman spectroscopy. Spectra were compared with biochemical analysis on the same samples which allowed identification of specific composition patterns in each tissue regardless of the cultivar. Considering the low penetration depth of ATR-FTIR signal, the cuticles were evidenced on the external sides of outer pericarp, hyaline layer, and testa. Spectra from testa of red and white wheats were clearly distinguished. FTIR spectroscopy, combined with statistical analysis, was successful in identifying the specific spectral signature for each peripheral tissue of wheat grains. In the 1500-800 cm(-1) spectral region, multivariate models allowed accurate prediction of the histological origin of the pericarp, hyaline, and aleurone layers regardless of the analyzed side, and the testa but with a lower performance