Health effects of β-glucan are typically related to dose, size and viscosity without taking the specific molecular structure into account. High β-glucan mutant barley, mother barley and oat β-glucans were large-scale extracted by comparable protocols using hot water, enzyme assisted hydrolysis and ethanol precipitation leading to similar molecular masses (200–300kDa). Multivariate data analysis on all compositional, structural and functional features demonstrated that the main variance among the samples was primarily explained by block structural differences as determined by HPSEC–PAD. In particular the barley high β-glucan mutant proved to exhibit a unique block structure with DP3 and DP4 contributions of: 78.9% and 16.7% as compared to the barley mother (72.1% and 21.4%) and oat (66.1% and 29.1%). This unique block structure was further confirmed by the 1 H NMR determination of the β-1,4 to β-1,3 linkage ratio. Low solubility of the barley samples was potentially an effect of substructures consisting of longer repetitive cellotriosyl sequences. FT-Raman and NMR spectroscopy were useful in measuring sample impurities of α-glucans and prediction of β-linkage characteristics.