Regional differences in tick-borne disease epidemiology may be related to biological variations between vector populations. Amblyomma aureolatum (Ixodida: Ixodidae), a neotropical tick, is known from several regions in Brazil. However, only in the metropolitan area of São Paulo (SP) state are there studies that establish its role as a vector of a pathogenic rickettsia (Rickettsia rickettsii). The aim of the study was to analyze the genetic diversity, population dynamics, and rickettsia infection in A. aureolatum populations from different spotted fever scenarios in Brazil. Samples were subjected to DNA extraction, amplification and sequencing of 12S rDNA, cytochrome oxidase subunit II and D-loop mitochondrial markers for tick population analyses, and gltA, htrA, ompA, and ompB genes for rickettsia researches. Of the 7–17 tick haplotypes identified, 5–13 were exclusive to each population and 2–12 for each epidemiological scenario, as well as three haplotypes shared by all populations. Amblyomma aureolatum populations are expanding, and do not appear to be genetically structured vis-a-vis the different epidemiological scenarios studied. Rickettsia bellii (in SP) and Rickettsia felis (in Santa Catarina) were identified as infecting A. aureolatum. No relationship between tick haplotypes and rickettsia types were observed.