Many organisms, such as insects, filarial nematodes, and ticks, contain heritable bacterial endosymbionts that are often closely related to transmissible tickborne pathogens. These intracellular bacteria are sometimes unique to the host species, presumably due to isolation and genetic drift. We used a polymerase chain reaction/electrospray ionization-mass spectrometry assay designed to detect a wide range of vectorborne microorganisms to characterize endosymbiont genetic signatures from Amblyomma americanum (L.), Amblyomma maculatum Koch, Dermacentor andersoni Stiles, Dermacentor occidentalis Marx, Dermacentor variabilis (Say), Ixodes scapularis Say, Ixodes pacificus Cooley & Kohls, Ixodes ricinus (L.), and Rhipicephalus sanguineus (Latreille) ticks collected at various sites and of different stages and both sexes. The assay combines the abilities to simultaneously detect pathogens and closely related endosymbionts and to identify tick species via characterization of their respective unique endosymbionts in a single test.