Amblyomma maculatum Koch (Acari: Ixodidae) is established in western Tennessee, a region with increased risk for Rocky Mountain spotted fever and ehrlichiosis. This tick transmits Rickettsia parkeri to humans, likely contributing to cases of rickettsiosis in the region. The objective was to determine pathogen associations within questing and host-collected A. maculatum, and identify ecological factors associated with pathogen infection that may increase the effectiveness of surveillance methods. Of 265 ticks tested, 60 (22.6%) were infected with R. parkeri, and 15 (5.7%) with Candidatus Rickettsia andeanae, a Rickettsia of unknown pathogenicity. Two deer-collected ticks tested positive for Ehrlichia ewingii. No ticks were positive for Anaplasma or Borrelia species. None of the ecological factors tested (collection month, collection source, sex, and habitat type) were associated with R. parkeri infection. This project developed baseline prevalence and incidence data for monitoring pathogen prevalence in A. maculatum populations, and identified an inexpensive method for distinguishing R. parkeri from Ca. R. andeanae.