Conjugative transfer and replacement of hundreds of kilobases of a bacterial chromosome can occur in vitro, but replacements in nature are either an order of magnitude smaller or involve the movement of mobile genetic elements. We discovered that two lineages of Staphylococcus aureus, including a pandemic methicillin-resistant lineage, were founded by single chromosomal replacements of at least ∼244 and ∼557 kb representing ∼10 and ∼20% of the chromosome, respectively, without the obvious involvement of mobile genetic elements. The replacements are unprecedented in natural populations of bacteria because of their large size and unique structure and may have a dramatic impact on bacterial evolution.