Cereal Chem. 85(4):571-577Temporal and genotypic differences in bulk carbohydrate accumulation in three barley genotypes differing in the content of mixed linkage β-(1→3),(1→4)-D-glucan (β-glucan) and starch were investigated using proton high-resolution, magic angle spinning, nuclear magnetic resonance ( 1 H HR MAS NMR) during grain filling. For the first time, 1 H HR MAS NMR spectra of flour from immature barley seeds are analyzed. Spectral assignments are made using two-dimensional (2D) NMR methods. Both αand β-glucan biosynthesis were characterized by inspection of the spectra as well as by calibration to the reference methods for starch and β-glucan content. Starch was quantified with very good calibrations to the α-(1→4) peak (5.29-5.40 ppm) and the region 3.67-3.83 ppm covering starch glycopyranosidic protons from H5 and H6. In contrast, the spectral inspection of the β-anomeric region 4.45-4.85 ppm showed unexpected lack of intensity in the high β-glucan mutant lys5f at seed maturity, resulting in poor calibration to reference β-glucan content. We hypothesize that the lack of β-glucan signal in lys5f indicates partial immobilization of the β-glucan that appears to be either genotypic dependent or water/β-glucan ratio dependent.