Due to an increasing diversity of bacterial pathogens known to be transmitted by hard ticks (Acari: Ixodidae) in North America, a comprehensive assay is needed to detect and differentiate among these numerous tick-borne pathogens. We describe an expanded protocol using a combination of multiplex polymerase chain reaction and reverse line blot hybridization to detect a greater diversity of infectious agents than were previously detectable. Ten novel oligonucleotide probes, either individually or in concert, enabled or enhanced identification of six Borrelia species, three Rickettsia species, and one Ehrlichia species. Simultaneous detection of these numerous tick-borne pathogens can advance surveillance efforts and improve accuracy of detection and, thus, reporting.