The use of whole-genome sequencing to monitor bacterial pathogens has provided crucial insights into their within-host evolution, revealing mutagenic and selective processes driving the emergence of antibiotic resistance, immune evasion phenotypes and adaptations that enable sustained human-to-human transmission. Deep genomic and metagenomic sequencing of intra-host pathogen populations is also enhancing our ability to track bacterial transmission, a key component of infection control. This Review discusses the major processes driving bacterial evolution within humans, including both pathogenic and commensal species. Initially, mutational processes, including how mutational signatures reveal pathogen biology, and the selective pressures driving evolution are considered. The dynamics of horizontal gene transfer and intra-host pathogen competition are also examined, followed by a focus on the emergence of bacterial pathogenesis. Finally, the Review focuses on the importance of within-host genetic diversity in tracking bacterial transmission and its implications for infectious disease control and public health.