The crossing of environmental barriers poses major adaptive challenges. Rareness of freshwater-marine transitions separates the bacterial communities, but how these are related to brackish counterparts remains elusive, as do the molecular adaptations facilitating cross-biome transitions. We conducted large-scale phylogenomic analysis of freshwater, brackish, and marine quality-filtered metagenome-assembled genomes (11,248). Average nucleotide identity analyses showed that bacterial species rarely existed in multiple biomes. In contrast, distinct brackish basins cohosted numerous species, but their intraspecific population structures displayed clear signs of geographic separation. We further identified the most recent cross-biome transitions, which were rare, ancient, and most commonly directed toward the brackish biome. Transitions were accompanied by systematic changes in amino acid composition and isoelectric point distributions of inferred proteomes, which evolved over millions of years, as well as convergent gains or losses of specific gene functions. Therefore, adaptive challenges entailing proteome reorganization and specific changes in gene content constrains the cross-biome transitions, resulting in species-level separation between aquatic biomes.