Fuels and chemicals derived through biochemical conversion of agricultural by-products such as wheat straw (Triticum aestivum L.) is an area currently under intense research. In this study, separate leaves and stems were hydrothermally pretreated and enzymatically hydrolysed and analysed chemically and by comprehensive microarray polymer profiling (CoMPP). This way, the effects of each degradation step to the intermolecular organisation of specific polysaccharides in the cell walls were elucidated. After pretreatment, the degree of polymerisation (DP) of released xylo-oligosaccharides in both samples was up to about 20, but mostly around 3–8, and notably more acetylated in stems. Arabinoxylan (AX) and mixed-linkage glucan (MLG) became water-extractable while xylan, xyloglucan (XG), mannan and glucan remained only alkali-extractable. All polysaccharides became partly digestible after pretreatment however, regardless their extractability in water or only alkali. Based on the results, AX and MLG appear to be loosely bound in the cell wall matrix while the other polysaccharides are bound more tightly and shielded from enzymatic attack by AX and MLG until pretreatment. The gradual solubilisation and digestion of the polysaccharides during pretreatment and hydrolysis correlate well with previous models of the polysaccharides’ structural organisation in the cell wall.