Equines play a role in the epidemiology of Brazilian spotted fever (BSF) since they are a primary host for the tick Amblyomma sculptum. We studied the seroprevalence for three species of Rickettsia in equines in four endemic (with human cases) and in four non-endemic areas (no human cases) in the Piracicaba River Basin, São Paulo, Brazil. A serological survey of 504 equines was performed: around 63 animals were sampled in each area and tested through indirect immunofluorescence assay for R. rickettsii, R. parkeri, and R. bellii in 2012-2013. Blood samples were seropositive for 183 equines (36.3%) in which 73 (39.9%) were from non-endemic areas. In the studied sites equines were highly exposed to Rickettsia infection ranging from 6.1% to 54.7%, with Geometric Mean Titers greater in endemic area (p = 0.012). Results suggest that Rickettsia may be more widespread than the surveillance of BSF has detected. These results highlight the need to include data on the seroprevalence of sentinel animals to improve human diagnoses and surveillance in areas with no reported human cases.