Rodents (87 Mus spretus and 10 Rattus norvegicus) collected in a suburban area of Seville (Andalusia, Spain) between September 2003 and April 2004 were tested by polymerase chain reaction for the presence of Bartonella DNA using primers amplifying a fragment of the 16S/23S rRNA internal transcribed spacer (ITS). Sequence analyses of the ITS of these Bartonella demonstrated that rodents in this region harbored at least three strains: two Bartonella genotypes detected in M. spretus, which clustered separately, and one genotype corresponding with B. tribocorum in R. norvegicus. The finding of Bartonella species in a high proportion of the rodent samples suggests the need to investigate whether these agents might be responsible for cases of febrile illnesses of unknown etiology in Southern Spain.