Media compositions affecting thermophilic biohydrogen production from waste glycerol were optimized using response surface methodology (RSM) with central composite design (CCD). Investigated parameters used were waste glycerol concentration, urea concentration, the amount of Endo-nutrient addition and disodium hydrogen phosphate (Na2HPO4) concentration. Waste glycerol concentration and the amount of Endo-nutrient addition had a significant individual effect on the cumulative hydrogen production (HP) (p <= 0.05). The interactive effect on HP was found between waste glycerol and urea concentration as well as waste glycerol concentration and the amount of Endo-nutrient addition (p <= 0.05). The optimal media compositions were 20.33 g/L of waste glycerol, 0.16 g/L of urea, 3.97 g/L of Na2HPO4 and 0.20 mL/L of the amount of Endo-nutrient addition which gave the maximum HP of 1470.19 mL H-2/L. The difference between observed HP (1502.84 mL H-2/L) and predicted HP was 2.22%. The metabolic products from the fermentation process were 1,3-propanediol (1,3-PD), ethanol, acetic, formic, lactic, butyric, and propionic acids. Results from polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis indicated that the hydrogen producers present in the fermentation broth was Thermoanaerobacterium sp. Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.