Ammonia oxidation in marine and estuarine sediments plays a pivotal role in the cycling and removal of nitrogen. Recent reports have shown that the newly discovered ammonia-oxidizing archaea can be both abundant and diverse in aquatic and terrestrial ecosystems. In this study, we examined the abundance and diversity of ammonia-oxidizing archaea (AOA) and betaproteobacteria (β-AOB) across physicochemical gradients in San Francisco Bay – the largest estuary on the west coast of the USA. In contrast to reports that AOA are far more abundant than β-AOB in both terrestrial and marine systems, our quantitative PCR estimates indicated that β-AOB amoA (encoding ammonia monooxygenase subunit A) copy numbers were greater than AOA amoA in most of the estuary. Ammonia-oxidizing archaea were only more pervasive than β-AOB in the low-salinity region of the estuary. Both AOA and β-AOB communities exhibited distinct spatial structure within the estuary. AOA amoA sequences from the north part of the estuary formed a large and distinct low-salinity phylogenetic group. The majority of the β-AOB sequences were closely related to other marine/estuarine Nitrosomonas-like and Nitrosospira-like sequences. Both ammonia-oxidizer community composition and abundance were strongly correlated with salinity. Ammonia-oxidizing enrichment cultures contained AOA and β-AOB amoA sequences with high similarity to environmental sequences. Overall, this study significantly enhances our understanding of estuarine ammonia-oxidizing microbial communities and highlights the environmental conditions and niches under which different AOA and β-AOB phylotypes may thrive.