Fleas, lice, and ticks collected in Peru in a suburban area of Cusco in November 1998 were tested by polymerase chain reaction for the presence of Bartonella DNA using primers amplifying a fragment of the intergenic spacer region (ITS) gene. Three new Bartonella genotypes were detected in Pulex fleas self-collected from the beds and clothes of schoolchildren and adults. A fourth new genotype was also detected from a tick found on a sheep in the same area. One of the genotypes is closely related to B. vinsoni subsp. berkhoffii, and the others seem to originate from unknown Bartonella species, whose medical importance has yet to be clarified.