The pan-Neisseria microarray was the first bacterial microarray to address multiple strains and species, and is a tool specifically developed for the performance of comparative studies within and between species. To achieve this, its design was based upon a detailed comparison of multiple genomes, prior to probe selection, and serial triage to optimize sensitivity and specificity. While this tool can be used for transcriptional comparisons of the same species, such as isogenic mutants, or strains exposed to different environmental conditions, its features are also particularly suited to population and functional studies of unrelated strains. The optimal use of these tools, including the use of single-channel labeling for genomic studies, the biological replication needed to perform robust transcription studies, and key aspects of data analysis such as the use of cross-channel correction and Bayesian analytical approaches, is discussed.