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A suitable pretreatment is a prerequisite of efficient acetone-butanol-ethanol (ABE) production from wood by Clostridia. In this study, organosolv fractionation, an effective pretreatment with ability to separate lignin as a co-product, was evaluated for ABE production from softwood pine and hardwood elm. ABE production from untreated woods was limited to the yield of 81 g ABE/kg wood and concentration of 5.5 g ABE/L. Thus, the woods were pretreated with aqueous ethanol at elevated temperatures before hydrolysis and fermentation to ABE by Clostridium acetobutylicum. Hydrolysis of pine and elm pretreated at 180 °C for 60 min resulted in the highest sugar concentrations of 16.8 and 23.2 g/L, respectively. The hydrolysate obtained from elm was fermented to ABE with the highest yield of 121.1 g/kg and concentration of 11.6 g/L. The maximum yield of 87.9 g/kg was obtained from pine pretreated for 30 min at 150 °C. Moreover, structural modifications in the woods were investigated and related to the improvements. The woody biomasses are suitable feedstocks for ABE production after the organosolv pretreatment. Effects of the pretreatment conditions on ABE production might be related to the reduced cellulose crystallinity, reduced lignin and hemicellulose content, and lower total phenolic compounds in the hydrolysates.