In this study, we investigate the isolation of lignin from the unhydrolyzed solids (UHS) by-product stream of 2nd generation biofuel production. Under reflux conditions, aqueous solutions of acetone, ethanol (EtOH), acetic acid (AcOH), and gamma-valerolactone (GVL) displayed approximately 53, 51, 53, and 65% yields of extractable solids, respectively, from corn stover UHS after Ammonia Fiber Expansion pretreatment and then enzymatic hydrolysis. Detailed chemical characterization was done including nuclear magnetic resonance, gel permeation chromatography, and thermogravimetric analysis. This characterization showed that material extracted from UHS using EtOH:H2O and acetone:H2O contained a lignin fraction that most resembled native lignin; although, the material extracted using acetone:H2O contained a significant carbohydrate component. Results suggest that solvent polarity, rather than other solvent properties (e.g., reflux temperature), are dominating factors determining the mass yields of extractable solids from UHS.