Fast growing hardwoods are one of the major renewable resources available to produce bio-based materials and green chemicals such as biofuel. However, the industrial processing of lignocellulosic biomass is hindered by the complex molecular structure and organization of the cell wall polysaccharides within the cell wall. This highlights the necessity of understanding the structural organization of cell wall polymers in industrially relevant tree species and improving green processing strategies to enhance biomass conversion to valuable products. In the present study, we implemented a hydrothermal step by subcritical water extraction (SWE) of non-cellulosic cell wall polymers in aspen wood prior to saccharification. Majority of pectin fraction was extracted within first 10 minutes of the SWE process while the extracts of consecutive 20- and 30-minute steps were enriched with acetylated xylan. Prolonged extraction resulted in increased lignin content and partial deacetylation of xylan. Oligomeric mass profiling of sequential extracts revealed that the glucuronic acid spacing pattern differed in an easily extractable versus recalcitrant glucuronoxylan populations. The electron microscopy analysis of the residues showed micromorphological changes on the fibre surface and cellulose macrofibrils. The analysis of saccharification potential without acid pretreatment revealed that the glucose yields from the residues after SWE treatment were significantly improved compared to that of aspen wood chips. Our study demonstrates the benefits of implementing a hemicellulose-first pre-treatment of aspen wood prior to saccharification to promote the simultaneous extraction of non-cellulosic cell wall polymers and reduce the requirement of acid pretreatment of biomass for bioethanol production.