The effect of hydrothermal pretreatment on chemical composition, microscopic structure and enzymatic digestibility of wheat straw was studied. Wheat straw was pretreated with increasing severity to obtain series of samples with altered chemistry and structure. The hydrothermal pretreatment caused solubilisation of arabinoxylan and phenolic acids and their dimers in a temperature dependent manner with minor effects on the cellulose and Klason lignin content. In the cell wall level, the pretreatment intensified staining of cellulose and relocalised xylan in the cell walls. The distribution, properties and content of the cell wall phenolic compounds was altered as observed with phloroglucinol and autofluorescence imaging. In the enzymatic hydrolysis, the highest yields were obtained from the samples with a low xylan and diferulate content. On the cell wall structural level, the sample with the highest digestibility was observed to have intensified cellulose staining, possibly reflecting the increased accessibility of cellulose.