Abstract Pathogens that are harmless in one environment can cause a serious disease in another. Among host-associated bacteria, transition between hosts can have serious consequences for animal and human health. However, much remains unknown about how adaptation shapes bacterial distribution in the wild. Here, investigating the ecological genomics of Escherichia coli from diverse hosts and environments, we address the idea that bacteria disperse freely, and challenge the “everything is everywhere” paradigm. Using comparative genomics and parallelised high throughout pangenome-wide association studies (900 experiments) we investigate lineage distribution and identify adaptive genomic signatures associated with host species, physiology and ecology. Our findings provide insights into bacterial niche adaptation, emphasize the impact of agriculture on microbial evolution, and inform One Health frameworks by linking genomics, host ecology, and the emergence of antimicrobial resistance.