The present study was performed in a non-endemic area for spotted fever (SF) in Imperatriz microregion, state of Maranhão, Brazil. Blood samples and ectoparasites were collected from 300 dogs of the Imperatriz microregion. Canine serum samples were tested individually by indirect immunofluorescence assay (IFA), using five Rickettsia isolates from Brazil. Antibodies reactive to at least one of the five species of Rickettsia were detected in 1.6% of the dogs (5/300). These sera were considered reactive to Rickettsia rickettsii and Rickettsia amblyommatis or very closely related species. The ticks (Acari: Ixodidae), identified as Rhipicephalus sanguineus sensu lato (Latreille), and the fleas, identified as Ctenocephalides felis, were tested by polymerase chain reaction (PCR) for detection of rickettsial DNA. More than 78% (83/106) of the C. felis fleas were found to be infected with Rickettsia species using gltA as rickettsial PCR targets, whereas no evidence of Rickettsia spp. was found in R. sanguineus s. l. Genetic analysis based on genes gltA, htrA and ompB showed that the detected strain, is most closely related to Rickettsia asembonensis (formerly Candidatus Rickettsia asemboensis). The present study is the first report of a R. asembonensis related infecting C. felis fleas in Brazil.