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Abstract 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 γ-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:H 2 O and acetone:H 2 O contained a lignin fraction that most resembled native lignin; although, the material extracted using acetone:H 2 O 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. Graphical abstract Image 1 Highlights • Polar and high reflux temperature solvents had the highest mass yields of extracted material from UHS. • The addition of water to polar organic solvents improved mass yields of extracted material from UHS. • Non-polar solvents were highly selective for lignin extraction from UHS. • Materials extracted using ethanol/water and acetone/water most resembled native lignin. [ABSTRACT FROM AUTHOR]