Sugar cane internodes have great morphological heterogeneity and different cell types that respond in varied ways to pretreatment processes. At the outermost region of the internode there are epithelial cells and a ring of cortical cells. Toward the central part of the internode there are a number of vascular bundles surrounded by parenchyma cells. The innermost regions are richer in parenchyma cells. The sugar cane internode regions were evaluated according to their response to, alkaline-sulfite pretreatment and the consequences for material recalcitrance. The experimental data set included 4 sugar cane hybrids dissected into 3 different internode regions that were pretreated for 5 different reaction times, totaling 60 samples. Cellular ultraviolet microspectrophotometric evaluation of the samples suggested that in the thin cell walls (parenchyma and vessels) the hydroxycinnamic acids were accessible to the pretreatment reagents, whereas in the thicker fiber cell walls lignin and hydroxycinnamic acids were more resistant. The outermost regions were the most resistant to lignin and hemicellulose removal. Enzymatic hydrolysis of the pretreated samples indicated that the outermost fraction and the rind were recalcitrant regions, whereas the pith rind interface was less recalcitrant. The parenchyma-rich pith rind interface region benefitted not only from delignification during pretreatment but also from hydroxycinnamic acid removal. By contrast, the most external fractions required a longer pretreatment time to overcome the recalcitrance because it was necessary to remove significant amounts of lignin and hydroxycinnamic acids from the samples.