An organosolv pretreatment consisting of an H 2 SO 4 /n-butanol biphasic system was designed to separate lignocellulosic biomass in three distinct phases: a cellulose-rich solid residue, hydrolyzed hemicelluloses in an aqueous phase, and lignin dissolved in a hydrophobic butanol phase. In the present study, the versatility of the process was investigated on materials of various compositions and origins: sugar cane bagasse, tall fescue, sugar beet pulp, eucalyptus, beech, and Japanese cedar. The efficiency was assessed in terms of lignin removal from the raw biomass and purity of the recovered cellulosic residue using the Klason method as well as improvement on enzymatic saccharification (increased from 18.7% to 96%). Results were correlated to biomass types and composition, and in comparison to an organic solvent-free method (dilute acid). Up to 81% cellulose purity corresponding to 87% lignin removal was achieved. Results were corroborated by scanning electron microscopy showing an absence of lignin deposition. Lignin molecular weight (GPC), structure (2D-HSQC NMR), recovery, and purity (up to 96%) have been investigated. Moreover, organic compounds responsible for fermentation inhibition were partially solubilized in the butanol, decreasing the concentration in the aqueous phase. Efficient butanol pretreatment applied on hardwood, bagasse, and herbaceous matter is promising. However, Japanese cedar (softwood) was too recalcitrant for this process.