Until recently, Amblyomma maculatum, (the Gulf Coast tick), has garnered little attention relative to other species of human-biting ticks in the United States. A. maculatum is now recognized as the principal vector of Rickettsia parkeri, a pathogenic spotted fever group rickettsia (SFGR) that causes an eschar-associated illness in humans that resembles Rocky Mountain spotted fever. A novel SFGR, distinct from other recognized Rickettsia spp., has also been detected recently in A. maculatum specimens collected from several regions of the southeastern United States. In this study, 198 questing adult Gulf Coast ticks were collected from 4 locations in Florida and Mississippi; 28% were infected with R. parkeri and 3% with a novel SFGR. Seventeen isolates of R. parkeri were cultivated in Vero E6 cells from individual specimens of A. maculatum; however, all attempts to isolate the novel SFGR were unsuccessful. Partial genetic characterization of the novel SFGR revealed identity with several recently described, incompletely characterized, and non-cultivated SFGR including Candidatus 'Rickettsia andeanae' and Rickettsia sp. 'Argentina', detected in several species of Neotropical ticks from Argentina and Peru. These findings suggest that each of these 'novel' rickettsiae represent the same species. Our study expands considerably the number of low-passage, A. maculatum-derived isolates of R. parkeri, and characterizes a second and sympatrically distributed Rickettsia sp. found in Gulf Coast ticks.