Because bacterial recombination involves the occasional transfer of small DNA fragments between strains, different sets of niche-specific genes may be maintained in populations that freely recombine at other loci. Therefore, genetic isolation may be established at different times for different chromosomal regions during speciation as recombination at niche-specific genes is curtailed. To test this model, we separated sequence divergence into rate and time components, revealing that different regions of the Escherichia coli and Salmonella enterica chromosomes diverged over a approximately 70-million-year period. Genetic isolation first occurred at regions carrying species-specific genes, indicating that physiological distinctiveness between the nascent Escherichia and Salmonella lineages was maintained for tens of millions of years before the complete genetic isolation of their chromosomes.