Microbes exhibit remarkably high genetic diversity compared with plant and animal species. Many phylogenetically diverse but apparently functionally redundant microbial taxa are detectable within a cubic centimetre of mud or a millilitre of water, and the significance of this diversity, in terms of ecosystem function, has been difficult to understand. Thus it is not known whether temporal and spatial differences in microbial community composition are linked to particular environmental factors or might modulate ecosystem response to environmental change. Fifty-three water and sediment samples from upper and lower Chesapeake Bay were analysed in triplicate arrays to determine temporal and spatial patterns and relationships between ammonia-oxidizing bacterial (AOB) communities and environmental variables. Thirty-three water samples (three depths) collected during April, August and October, 2001–2004, from the oligohaline upper region of the Bay were analysed to investigate temporal patterns in archetype distribution. Using a combination of a non-weighted discrimination analysis and principal components analysis of community composition data obtained from functional gene microarrays, it was found that co-varying AOB assemblages reoccurred seasonally in concert with specific environmental conditions, potentially revealing patterns of niche differentiation. Among the most notable patterns were correlations of AOB archetypes with temperature, DON and ammonium concentrations. Different AOB archetypes were more prevalent at certain times of the year, e.g. some were more abundant every autumn and others every spring. This data set documents the successional return to an indigenous community following massive perturbation (hurricane induced flooding) as well as the seasonal reoccurrence of specific lineages, identified by key functional genes, associated with the biogeochemically important process nitrification.