The performance of biohydrogen production in an anaerobic sequencing batch reactor (ASBR) was evaluated with respect to variations in the key operational parameters pH, hydraulic retention time HRT, and organic loading rate OLR using sugar refinery wastewater as substrate. Analysis of variance (ANOVA) indicated HRT had less significant influence on hydrogen content and yield in comparison to pH and OLR, whereas OLR has much impact on hydrogen production rate. Taxonomic analysis results showed that diverse bacterial species contributed to hydrogen production and the dominant species in the bioreactor were governed by all operational parameters. Even without pretreatment of the seed sludge, a high proportion of Clostridium spp. over the other bacterial species was observed at pH 5.5, and this is compatible with the high hydrogen productivity. Consequently, pH 5.5, HRT 10 h, and OLR 15 kg/m(3) d were delineated as the optimal operational conditions for an ASBR fed with sugar refinery wastewater. Copyright (C) 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.