Rickettsial diseases, such as Rocky Mountain spotted fever, pose a public health threat because of humans' interrelationship with common arthropod species, such as ticks, mites, fleas, and lice. Individuals may come in contact with these vectors of disease on a fairly regular basis either directly or indirectly through pets or wildlife species, at home or in recreational areas. Therefore, it is of vital importance to know and understand the geographical distribution and prevalence of disease and rickettsial-infected arthropods. We analyzed Dermacentor variabilis ticks from nature found positive for Rickettsia sp. to determine the specific species present. Rickettsiae were detected through a 17-kDa surface antigen seminested PCR. Seminested PCR represents a sensitive and specific molecular technique in which to identify the presence of bacteria within arthropod hosts. Through sequence analysis of this gene, three Rickettsia species, Rickettsia bellii , Rickettsia montanensis , and Rickettsia rickettsii , were detected in a single tick specimen. Further molecular analyses of the 17-kDa surface antigen and the citrate synthase gene were also performed to support this finding. This is the first report of the detection of multiple Rickettsia species from a single D. variabilis tick in nature.