Evidence of spotted fever group (SFG) rickettsiae was obtained from flea pools and individual ticks collected at three sites in northwestern Peru within the focus of an outbreak of febrile disease in humans attributed, in part, to SFG rickettsia infections. Molecular identification of the etiologic agents from these samples was determined after partial sequencing of the 17-kDa common antigen gene (htrA) as well as pairwise nucleotide sequence homology with one or more of the following genes: gltA, ompA, and ompB. Amplification and sequencing of portions of the htrA and ompA genes in pooled samples (2 of 59) taken from fleas identified the pathogen Rickettsia felis. Four tick samples yielded molecular evidence of SFG rickettsiae. Fragments of the ompA (540-bp) and ompB (2,484-bp) genes were amplified from a single Amblyomma maculatum tick (tick 124) and an Ixodes boliviensis tick (tick 163). The phylogenetic relationships between the rickettsiae in these samples and other rickettsiae were determined after comparison of their ompB sequences by the neighbor-joining method. The dendrograms generated showed that the isolates exhibited close homology (97%) to R. aeschlimannii and R. rhipicephali. Significant bootstrap values supported clustering adjacent to this nodule of the SFG rickettsiae. While the agents identified in the flea and tick samples have not been linked to human cases in the area, these results demonstrate for the first time that at least two SFG rickettsia agents were circulating in northern Peru at the time of the outbreak. Furthermore, molecular analysis of sequences derived from the two separate species of hard ticks identified a possibly novel member of the SFG rickettsiae.Proteobacteria of the family Rickettsiaceae, order Rickettsiales, are made up of highly specialized obligate intracellular, gram-negative bacteria that survive freely within the cytosol of the host cell. Members of the genus Rickettsia are divided into two genetically similar groups, the spotted fever group (SFG) and the typhus group (TG), on the basis of host specificity, intracellular location, in vitro growth conditions, antigenic characteristics, the molecular sequences of conserved genes, clinical features, and epidemiology (15,16,37,38). Seventeen species of the genus Rickettsia are categorized within the SFG rickettsiae. With the exception of Rickettsia akari (mite-borne) and R. felis (flea-borne), the remaining SFG rickettsia species are recognized as tick-borne rickettsiae that are passed to subsequent generations or stages transovarially and transtadially (21). While the members of the SFG rickettsiae are adapted to existence within specific hosts, they are capable of infecting humans after humans are bitten by infected arthropods. The TG contains two species, R. prowazekii and R. typhi.