A thermotolerant Klebsiella sp. TR17 for production of hydrogen from crude glycerol was investigated. Results from Plackett-Burman design indicated that the significant variables, which influenced hydrogen production, were KH2PO4 and NH4Cl (for buffer capacity and nitrogen source). Subsequently, the two selected variables and crude glycerol were optimized by the Central Composite design for achieving maximum hydrogen and ethanol yield. The concentration of crude glycerol, KH2PO4, and NH4Cl had an individual effect on both hydrogen and ethanol yield (p < 0.05), while KH2PO4 and NH4Cl had an interactive effect on ethanol yield (p < 0.05). The optimum medium components were 11.14 g/L crude glycerol, 2.47 g/L KH2PO4, and 6.03 g/L NH4Cl. The predicted maximum simultaneous hydrogen and ethanol yield were 0.27 mol H-2/mol glycerol and 0.63 mol EtOH/mol glycerol, respectively. Validation of the predicted optimal conditions exhibited similar hydrogen and ethanol yield of 0.26 mol H-2/mol glycerol and 0.58 mol EtOH/mol glycerol, respectively. Copyright (C) 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.