Hydrogen-producing microorganisms are believed to play an important role in energy metabolism of micro-organisms in anaerobic environments and hence are one of the crucial factors for influencing the activity and develop-ment of these microorganisms. Consequently, they provide the biological foundation for the biotechnology such as MEOR (Microbial Enhanced Oil Recovery) and microbial fixation of CO2 and conversion of it into CH4 and etc. How-ever, knowledge on the community of hydrogen-producing microorganisms and their potential in subsurface formations are still limited. In this study, hydrogen-producing microorganisms in the production water from an oilfield as well as enrichment cultures were analyzed with clone library analysis of [FeFe]-hydrogenase encoding genes. The results show that [FeFe]-hydrogenase genes in production water are diverse and related to Bacteroidetes, Firmicutes, Spirochaetes and uncultured. Anaerobic incubations established within the oil reservoir production water and generating 202 mmol H2/mol glucose during 7-day incubation at 55°C indicate a high frequency of members of the Firmicutes. This study implies that hydrogen-producing microorganisms in oil reservoir may play a positive role in promoting the in situ bio-process via hydrogen production once common nutrients are available. These data are helpful for evaluating, developing, and utilizing hydrogen-producing microorganisms in oil reservoirs for biological fixation and conversion of CO2 into CH4 as well as MEOR.