Eighty-nine Rhipicephalus sanguineus ticks and 21 Rhipicephalus bursa ticks collected in Catalonia were tested by the hemolymph test to establish their infection rate with spotted fever group rickettsiae. By Giménez staining, 11.2% of the R. sanguineus isolates and 0% of the R. bursa isolates were found to contain rickettsia-like organisms. Six spotted fever group rickettsial strains (Bar29, Bar31, Gir4, Tar1, Tar2, and Tar3) were isolated from these ticks and were characterized by phenotypic and genotypic analyses. PCR followed by restriction fragment length polymorphism analysis showed that the six strains were identical and were characterized by the same restriction profiles as a strain, Mtu5, previously isolated from Rhipicephalus turanicus ticks in the South of France. Microimmunofluorescence serotyping, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified organisms, and Western blot (immunoblot) assay with mouse polyclonal sera confirmed this observation. Pulsed field gel electrophoresis of the whole genome of three of the strains showed that, although closely related, the profile of Tar1 was slightly different from that of the Bar strains. Phylogenetic analysis showed that this new rickettsial sero- and genotype, which will be named the "Catalan strain," is closely related to Rickettsia massiliae. This strain shows an unexpected resistance to rifampin. The epidemiological implications of these findings are considered.