Like other aromatic hydrocarbons, benzene is a common soil and groundwater contaminant. It is recognized as a human carcinogen. Exposure of benzene can cause serious negative impacts on human health. Benzene is of major concern due to its toxicity and relatively high water solubility. Benzene is easily biodegraded by ubiquitous bacteria with the presence of free oxygen. However, soil and groundwater contamination with petroleum hydrocarbon often results in the development of anaerobic zones. Bioremediation has been considered as an advantageous alternative in terms of fairly low cost, process flexibility, and on-site utility for the treatment of contaminated soil and groundwater. However, benzene is particularly persistent under anaerobic condition even in the enhanced anaerobic biodegradation process. Although studies have shown that benzene biodegradation could occur under several reducing conditions, the in situ activities of anaerobic benzene degradation are generally low. Bioaugmentation rather than biostimulation may be applicable to accelerate biodegradation process. Successful bioaugmentation requires the inoculation of contaminated soil and groundwater with the strains or consortia of specific degrading capabilities. However, information of dominant species within the microorganisms for anaerobic benzene degradation is still limited. To address this problem, in this study, a benzene-degrading nitrate-reducing culture was established with soil contaminated by gasoline. A nitrate-reducing medium with sulphate, phosphate and other inorganic nutrient was employed to enhance anaerobic benzene degradation. BioSep BioTrap coupled with stable isotope probing and other molecular biological methods were used to identify key anaerobic benzene degraders. Members of genus Dokdonella spp., Pusillimonas spp., and Advenella spp. were found to be the dominant microorganisms during anaerobic benzene degradation, and were hypothesized to be benzene degrader under nitrate-reducing condition.