Methods linking environmental function and microbial community composition are limited by phylogenetic resolution and throughput time. Denaturing gradient gel electrophoresis (DGGE) allows for resolution of ammonia-oxidizing bacteria strains separated by as little as one base pair (bp) in 16S rRNA gene sequence but is low throughput. In contrast, microarrays are high throughput. We have designed a microarray platform to identify the majority of environmental strains of betaproteobacterial ammonia oxidizers using signature oligonucleotide sequences. Seventy-seven target sequences or single-nucleotide polymorphisms (SNP) were derived from 183 betaproteobacterial ammonia oxidizer 16S rRNA gene sequences representing the genera Nitrosospira and Nitrosomonas of the β sub-class of Proteobacteria and the cluster groups of strains within each genera identified using DNA homology values. These sequences were obtained from the National Center for Biotechnology Information database. Templates isolated from pure cultures of betaproteobacterial ammonia oxidizers and soil samples incubated with and without dairy slurry were amplified using primers CTO189f and CTO654r. Amplicons were analyzed via DGGE and microarray hybridization. Mixtures of pure culture strains of betaproteobacterial ammonia oxidizers were used to simulate environmental samples containing a known number of sequences and template concentrations. Our DNA oligonucleotide microarray demonstrated highly specific detection of individual betaproteobacterial ammonia oxidizer strains amplified from pure cultures, model communities, and soil samples and provided resolution of community composition comparable to that of DGGE. High-throughput analyses of betaproteobacterial ammonia oxidizer communities will further our knowledge of their ecology and link process level functions to management practices such as dairy slurry applications and specific strains of betaproteobacterial ammonia-oxidizing bacteria.