The microbial community structure of thermophilic mixed culture sludge used for bio-hydrogen production from palm oil mill effluent was analyzed by fluorescence in situ hybridization (FISH) and 165 rRNA gene clone library techniques. The hydrogen-producing bacteria were isolated and their ability to produce hydrogen was confirmed. The microbial community was dominated by Thermoanaerobacterium species (similar to 66%). The remaining microorganisms belonged to Clostridium and Desulfotomaculum spp. (similar to 28% and similar to 6%, respectively). Three hydrogen-producing strains, namely HPB-1, HPB-2, and HPB-3, were isolated. 16S rRNA gene sequence analysis of HPB-1 and HPB-2 revealed a high similarity to Thermoanaerobacterium thermosaccharolyticum (98.6% and 99.0%, respectively). The Therrnoanaerobacterium HPB-2 strain was a promising candidate for thermophilic fermentative hydrogen production with a hydrogen yield of 2.53 mol H-2 mol(-1)hexose from organic waste and wastewater containing a mixture of hexose and pentose sugars. Thermoanaerobacterium species play a major role in thermophilic hydrogen production as confirmed both by molecular and cultivation-based analyses. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.