During field work in Nazaré Paulista, state of São Paulo, Brazil, we found 13 (56.5%) of 23 birds (mostly Passeriformes) to be infested by 28 larvae and 1 nymph of Amblyomma spp. Two larvae were reared to the adult stage, being taxonomically identified as Amblyomma parkeri Fonseca and Aragão, whereas five larvae and one nymph were identified as Amblyomma longirostre Koch. All six A. longirostre specimens were shown to be infected by rickettsia, as demonstrated by polymerase chain reaction (PCR) targeting two rickettsial genes (gltA and ompA) or isolation of rickettsia in cell culture from one of the ticks. This isolate was designated as strain AL, which was established in Vero cell culture and was molecularly characterized by DNA sequencing fragments of the rickettsial genes gltA, htrA, ompA, and ompB. Phylogenetic analyses inferred from ompA and ompB partial sequences showed a high degree of similarity of strain AL with Rickettsia sp. strain ARANHA, previously detected by PCR in A. longirostre ticks from Rondônia, northern Brazil. We conclude that strain AL is a new rickettsia genotype belonging to the same species of strain ARANHA, which are closely related to Candidatus ’R. amblyommii’. Further studies should elucidate if strains AL and ARANHA are different strains of Candidatus ’R. amblyommii’ or are a new species.