Palm oil mill effluent (POME), an agro-industrial wastewater with high solids content, was subject to hydrolysis by 1% (w/v) nitric acid in order to increase its solubility and the fermentable sugar content from its cellulosic component. POME hydrolysate was then evaluated in an up-flow anaerobic sludge blanket (UASB) bioreactor for the production of biohydrogen gas via mixed culture under thermophilic conditions. The bioreactor was fed with pre-treated POME under varied hydraulic retention time (HRT) between 48 and 3 hat constant cycle length of 24 h to test the productivity of H-2 and the stability of UASB; no washout of biomass occurred at any cycle and the system managed to recover its H-2 production rate (HPR) after initial fluctuations. In this study, H-2-producing granules (HPGs) were formed shortly after the start-up period, and were analysed by FESEM, FTIR, SEM-EDX, and their extracellular polymeric substances (EPS) content. The maximum HY and HPR achieved were 2.45 mol-H-2/mol-sugar and 11.75 L-H2/L-POME d(-1), respectively, at HRT 6 h. Acetic acid was found to be the major by-product at all HRTs, followed by butyric acid, while Clostridium spp. was found to be the most dominant H-2-producing bacteria in the system. Results suggest that UASB has a good potential for stable H-2 production with high POME digestion rate. (C) 2018 Elsevier Ltd. All rights reserved.