In microbial fuel cells (MFC), wastewater is used as a fuel while organic and nutrient pollution in the wastewater are being treated. In the present study, commonly existing microbial populations in MFC anode biofilms were identified using high throughput FLX Titanium pyrosequencing to provide much more extensive information of anode microbial communities than previously possible. Using 454 FLX Titanium pyrosequencing, 31,901 sequence reads with an average length of 430 bp were obtained from 16S rRNA gene amplicons from different MFC anodes with different substrate exposure and respiration conditions, and microbial community structure and population identification were then analyzed using high-throughput bioinformatics methods. Although community profiles from the four samples were significantly different, hierarchical clustering analysis revealed several bacterial populations that commonly exist in the anode biofilm samples. These bacteria were phylogenetically distributed in Firmicutes and the alpha-, beta-, gamma-, and delta-subclasses of Proteobacteria. In addition, most of these populations were found to be novel anode bacteria and exhibited oligotrophic or substrate-concentration-insensitive growth. These findings suggest that commonly existing anode bacteria may play a key role in the stable operations of MFCs, combined with wastewater treatment plants, under fluctuating substrate and respiration conditions.