The insertion sequence IS629, which is highly prevalent in Escherichia coli O157:H7 genomes, was found to be absent in O157:H- strains, which are on a divergent pathway in the emergence of O157:H7. Although O157:H- is deficient in IS629, it permits IS629 transposition, with an excision frequency higher than that of ancestral O55:H7 strains but significantly lower than that of pathogenic O157:H7 strains.