Acidic ethanol organosolv fractionation of hybrid poplar was investigated to determine the impact of pretreatment conditions on the resulting biomass and lignin properties and to assess the subsequent deconstruction of the cell wall biopolymers to monomeric sugars and aromatics. It was found that increasing reaction severity (i.e., time and temperature) during the organosolv fractionation increased the rate of delignification and xylan solubilization while the lignins recovered from the liquors were found to exhibit lower degrees of polymerization. Glucose hydrolysis yields > 75% at moderate enzyme loadings (30 mg/g glucan) could be obtained for the more severe pretreatment conditions. The lignins recovered from the pretreatment liquors were subjected to fractionation using a sequential extraction with solvents of increasing polarity. It was found that the low molar mass, low polydispersity lignins increased in pretreatment liquors with increasing time and temperature and were concentrated in the methanol fraction while a high molar mass fraction was extracted with the diethyl ether. We hypothesize that the extraction of the high molar mass fraction with diethyl ether is due to partial ethyl O-alkylation of lignin hydroxyl groups during pretreatment, rendering lignins more soluble in the non-polar solvent. Finally, depolymerization of unfractionated lignins by thioacidolysis resulted in mass yields of aromatic monomers ranging from 80 to 157 mg monomer per gram of lignin and that these yields exhibited strong positive correlations to the lignin β-O-4 content, molar mass, and strong negative correlations to the pretreatment temperature.