Mountain pine beetle killed lodgepole pine (Pinus contorta) chips were pretreated by the ethanol organosolv process and the cellulose rich insoluble component was subsequently enzymatically hydrolyzed. The effect of varying pretreatment chemicals on the resulting substrate’s ease of enzymatic hydrolysis and physicochemical characteristics was investigated. It was apparent that the different pretreatment chemicals resulted in variations in the chemical composition of the solid and liquid fraction as well as in the extent of cellulolytic hydrolysis. Pretreatment under acidic conditions resulted in substrates that were readily hydrolyzed despite the observed increase in delignification selectivity obtained under alkaline conditions. Furthermore, acidic pretreatments also resulted in a lower cellulose degree of polymerization, shorter fiber lengths and increased substrate porosity. These results strongly suggest that a substrate’s susceptibility to enzymatic hydrolysis is influenced by its physical properties (especially enzyme accessible surface area) rather than its chemical composition.