This is the first report on the characterization of the microbial biofilm community structure and water quality change along a closed and stable raw water distribution system. 454-pyrosequencing was employed to investigate the microbial communities in four biofilm samples. 25,426 optimized sequences were obtained. Results showed Proteobacteria was the dominant phylum in each biofilm sample. The abundance of Nitrospiraes in M6 biofilm, Firmicutes in M8 biofilm, Actinobacteria in M9 biofilm were higher by comparing with other three biofilm samples. The M6 microbial biofilm community structure was similar to that of M7, dissimilar to that of M9. Dissolved oxygen and nitrogen was probably major factors to influence the microbial biofilm communities. Nitrospiraes in M6 biofilm and Firmicutes in M8 biofilm were crucial to remove ammonia nitrogen and nitrate in raw water. How to enrich functional microbes in biofilm to pretreat raw water is an important area of future research. (C) 2013 Elsevier Ltd. All rights reserved.