The binding of protein to DNA is central to the regulation of gene expression and the organization of chromosomal DNA. To date, there exist few techniques for the determination of genome-wide protein binding in prokaryotes, and none that are simultaneously simple, high-resolution, and rapid. I describe XATAC-seq, an adaptation of the eukaryotic assay for transposase-accessible chromatin with sequencing (ATAC-seq), combining formaldehyde crosslinking of DNA-protein complexes, adapter-loaded transposase treatment for next-generation sequencing library generation, and high-throughput sequencing to interrogate these genome-wide binding patterns in bacteria. The technique captures the binding of both major classes of prokaryotic DNA-binding proteins–transcription factors and nucleoid-associated proteins–genome-wide at the resolution of individual binding sites. XATAC-seq was applied to determine the protein occupancy landscapes of several bacterial species. Remarkably, the landscapes show a high degree of fidelity to specific nucleoid-associated proteins and demonstrate several conserved characteristics, including extended domains of high enrichment and preferential enrichment of AT-rich regions. This has led to the speculation that these nucleoid-associated proteins are members of a common high-level functional group, and that this class of nucleoid-associated protein is prevalent among a significantly wider range of prokaryotes than previously realized. In particular, the Mga protein of Streptococcus pyogenes is proposed to serve the high-level function of suppression of ectopic expression in an analogous fashion to the H-NS protein in E. coli. This work represents the first assessment of protein occupancy landscapes in gram-positive bacteria and a significant technical improvement over existing techniques for assaying genome-wide protein binding in prokaryotes.