Microbial community structure of acidogenic biofilm from long-term operated sequencing batch bioreactor producing biohydrogen was analyzed through culture independent technique. Bioreactor was operated under variable operation and substrate conditions for a period of 1435 days. Phylogenetic distribution showed a significant diversity and illustrated the presence of four dominant operational taxonomic units (OTUs) viz., Bacteroidia, Bacilli, Clostridia, Flavobacteria and Aquificae. Dominance of Clostridia and Bacilli classes were observed each with four OTUs. Majority of OTUs were found to produce fermentative H-2. Even at higher load and under diverse operating conditions bioreactor functioned without any process inhibition which indicates the robustness of sustained microbial community. Community structure of bioreactor was comparatively evaluated with other bioreactor producing H-2, operated with same parent culture and conditions but with different substrates, established the dynamics and shift of microbial diversity which corresponded to diverse substrates used for the bioreactor operation. Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.