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A total of 130 adult free-living ticks of Amblyomma cajennenseand 9 free-living Amblyomma dubitatumwere collected in the surroundings of the Pampulha Lake, within Belo Horizonte city, state of Minas Gerais, south-eastern Brazil. Each adult tick was tested for rickettsial infection by PCR protocols targeting the rickettsial genes gltA, htrA, and ompA. All the 130 A. cajennenseticks were negative by PCR. In contrast, all 9 A. dubitatumticks were shown to contain rickettsial DNA. PCR products were sequenced, generating identical sequences for each gene among the 9 ticks, which were shown to contain a novel agent, here designated as Rickettsiasp. strain Pampulha. Phylogenetic analyses inferred from the 3 rickettsial genes (gltA, htrA, ompA) showed that Rickettsiasp. strain Pampulha segregated in the same cluster with R. tamurae, R. monacensis, and several other unclassified rickettsial strains; however, strain Pampulha is a unique Rickettsiastrain for the New World, since its closest relatives, according to 3 genes (gltA, htrA, and ompA), are all from the Old World. Because A. dubitatumeventually bites humans, further studies are necessary to determine the potential pathogenicity of Rickettsiasp. strain Pampulha to humans.