Arabinoxylans (AX) and (1 -> 3),(1 -> 4)-beta-glucans (BG) are the main components of,cereal cell walls and influence many aspects of their end uses. Important variations in the composition and structure of these polysaccharides have been reported among cereals and cultivars of a given, species. In this work, the spatial distribution of AX and BG in the endosperm of mature grains was established for nine wheat varieties and eight barley varieties using enzymatically assisted mass spectrometry imaging (MSI). Importantstructural features of the AX and BG polymers that were previously shown to influence their physicochemical properties were assessed. Differences in the distribution of AX and BG structures were observed, both within the endosperm of a given cultivar and between wheat and barley cultivars. This study provides a unique picture of the structural heterogeneity of AX and BG polysaccharides at the scale of the whole endosperm in a series of wheat and barley cultivars. Thus, it can participate meaningfully in a strategy aiming at understanding the structure function relationships of these two polymers.