Obligately intracellular rickettsiae cause a broad spectrum of disease in humans and animals, ranging from mild illness to life-threatening infections. Multiple species co-circulate along the southern United States of America–northern Mexico border, yet their seroprevalence in susceptible hosts remains incompletely understood. Dogs serve as key amplifying hosts for several of these pathogens, including Rickettsia rickettsii and Rickettsia massiliae, and have been shown to be infected by flea-borne Rickettsia typhi and Rickettsia felis. To better characterize exposure and potential co-infection patterns, we conducted a large binational seroepidemiologic study of 779 dogs from urban households and shelter settings across seven subregions along both sides of the border. Using a custom multiplex micro-immunofluorescence assay, we simultaneously screened for antibodies to R. rickettsii, R. massiliae, R. typhi, and R. felis.