Background. Three distinctly different lineages of head and body lice are known to parasitize humans. One lineage includes head and body lice and is currently worldwide in distribution (type A). The other 2 (types B and C) include only head lice and are geographically restricted. It was hypothesized that head louse phylotypes were exchanged only recently, after European exploration and colonization (after Columbus). Methods. To determine which louse type or types were found in the Americas before European colonization, we used polymerase chain reaction in 2 laboratories to amplify DNA from 2 genes (Cytb and Cox1) belonging to 1000-year-old lice collected from Peruvian mummies. Results. Only the worldwide type (type A) was found. Therefore, this phylotype was worldwide before European colonization, as type A lice were common in Europe, Africa, and Asia. Conclusions. The findings of this study show that several phylotypes of head lice have coexisted for centuries in humans and support the claim that type A lice were present in the Americas before the time of Columbus. © 2008 by the Infectious Diseases Society of America. All rights reserved.