Molecular method such as PCR-DGGE using well-accepted universal 16S rDNA PCR primer sets was often applied on the study of Clostridium bacterial community. However, unsatisfied results were often obtained due to the difficulty of distinguishing coexisting Clostridium and other anaerobic microorganisms. In this study, a specific PCR primer set (Chis150f-ClostIr), based on the available rRNA gene sequences from the database, targeting only the majority of Clusters I and II Clostridia was designed and tested on both the pure culture Clostridium and dark fermentation sludge. it was demonstrated that this new primer set could not only successfully distinguish the coexisted Clostridium species but also revealed the existence of some Clostridium species in the sludge which could not be detected by using the universal primer set. This method successfully provides a detailed view on the Clostridium community responsible for an effective hydrogen production in dark fermentation system. (c) 2007 Published by Elsevier Ltd. on behalf of International Association for Hydrogen Energy.