| Summary: | Relapsing fever (RF) is an arthropod-borne disease caused by <i>Borrelia</i> spirochete, which is one of the major public health concerns in endemic regions including Africa. However, information on <i>Borrelia spirochetes</i> is limited in Zambia. Here, we investigate the <i>Borrelia spirochetes</i> harbored by <i>Ornithodoros</i> ticks in Zambian National Parks. We analyzed 182 DNA samples pooled from 886 <i>Ornithodoros</i> ticks. Of these, 43 tested positive, and their sequence revealed that the ticks harbored both Old and New World RF borreliae. This research presents the first evidence of Old-World RF borreliae in Zambia. The New World RF borreliae detected herein differed from the <i>Candidatus</i> Borrelia fainii previously reported in Zambia and were closely related to the pathogenic <i>Borrelia</i> sp. VS4 identified in Tanzania. Additionally, <i>Borrelia theileri</i> was recently reported in Zambia. Hence, at least four different <i>Borrelia</i> species occur in Zambia, and the organisms causing relapsing fever there might be more complex than previously thought. We empirically confirmed that real-time PCR with TaqMan minor groove binder probes accurately and simultaneously detected both Old and New World RF. In this manner, they could facilitate quantitative analyses of both types of RF borreliae. Subsequent investigations should endeavor to isolate the aforementioned <i>Borrelia</i> spp. and perform serosurveys on patients with RF.
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