Grasses are lignocellulosic materials useful to supply the billion-tons annual requirement for renewable resources that aim to produce transportation fuels and a variety of chemicals. However, the polysaccharides contained in grass cell walls are built in a recalcitrant composite. Deconstruction of these cell walls is still a challenge for the energy-efficient and economically viable transformation of lignocellulosic materials. The varied tissue-specific distribution of cell wall components adds complexity to the origins of cell wall recalcitrance in grasses. This complexity usually led to empirically developed pretreatment processes to overcome recalcitrance. A further complication is that efficient pretreatment procedures generally treat the less recalcitrant tissues more than necessary, which results in the generation of undesirable biomass degradation products.Six different sugarcane hybrids were used as model grasses to evaluate the tissue-specific distribution of hemicelluloses and the role of these components in cell wall recalcitrance. Acetylated glucuronoarabinoxylan (GAX) occurs in all tissues. Mixed-linkage glucan (MLG) was relevant in the innermost regions of the sugarcane internodes (up to 15.4 % w/w), especially in the low-lignin content hybrids. Immunofluorescence microscopy showed that xylans predominated in vascular bundles, whereas MLG occurred mostly in the parenchyma cell walls from the pith region of the hybrids with low-lignin content. Evaluation of the digestibility of sugarcane polysaccharides by commercial enzymes indicated that the cell wall recalcitrance varied considerably along the internode regions and in the sugarcane hybrids. Pith regions of the hybrids with high MLG and low-lignin contents reached up to 85 % cellulose conversion after 72 h of hydrolysis, without any pretreatment.The collective characteristics of the internode regions were related to the varied recalcitrance found in the samples. Components such as lignin and GAX were critical for the increased recalcitrance, but low cellulose crystallinity index, high MLG contents, and highly substituted GAX contributed to the generation of a less recalcitrant material.