The aim of this study was to promote biohydrogen production in an thermophilic anaerobic fluidized bed reactor (AFBR) at 55 degrees C using a mixture of sugar cane stillage and glucose at approximately 5000-5300 mg COD L-1. During a reduction in the hydraulic retention time (HRT) from 8, 6, 4, 2 and 1 h, H-2 yields of 5.73 mmol g CODadded-1 were achieved (at HRT of 4 h, with organic loading rate of 52.7 kg COD m(-3) d(-1)). The maximum volumetric H-2 production of 0.78 L H-2 h(-1) L-1 was achieved using stillage as carbon source. In all operational phases, the H-2 average content in the biogas was between 31.4 and 52.0%. Butyric fermentation was the predominant metabolic pathway. The microbial community in accordance with the DGGE bands profile was found similarity coefficient between 91 and 95% without significant changes in bacterial populations after co-substrate removal. Bacteria like Thermoanaerobacteriurn sp. and Clostridium sp. were identified. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.