Controlling microbial contamination of drinking water is critical to public health. However, understanding of the microbial ecology of drinking water remains incomplete. Representing the first application of high-throughput sequencing in drinking water microbiology, the objective of this study is to evaluate pyrosequencing as a high-throughput technique for the characterization of bacterial diversity in drinking water in comparison with conventional clone library analysis. Pyrosequencing and clone library analysis were performed in parallel to study the bacterial community composition in drinking water samples following the concentration of microbial biomass in drinking water with ultrafiltration. Validated by clone library analysis, pyrosequencing was confirmed as a highly efficient deep-sequencing technique to characterize the bacterial diversity in drinking water. Sequences of Alphaproteobacteria and Betaproteobacteria dominated the bacterial community in drinking water with Oxalobacteraceae and Methylobacteriaceae as the most abundant bacterial families, which is consistent with the prominent abundance of these populations frequently detected in various freshwater environments where source waters originate. Bacterial populations represented by the most abundant sequences in drinking water were closely related to cultures of metabolically versatile bacterial taxa widely distributed in the environment, suggesting a potential link between environmental distribution, metabolic characteristics, and abundance in drinking water.