The red wolf (Canis rufus) is a critically endangered North American canid, with surviving conspecifics divided between a captive breeding population and a reintroduced free-ranging population. The goal of this study was to assess the prevalence of selected vector-borne pathogens in captive red wolves. Whole blood samples were collected from 35 captive red wolves. Quantitative polymerase chain reaction (PCR) assays were performed on extracted DNA to identify infection by Trypanosoma cruzi and vector-borne organisms within the following genera: Anaplasma, Babesia, Bartonella, Ehrlichia, Mycoplasma, Neoehrlichia, Neorickettsia, and Rickettsia. All red wolves sampled were PCR-negative for all tested organisms. These pathogens are unlikely to constitute threats to red wolf conservation and breeding efforts under current captive management conditions. The results of this study establish a baseline that may facilitate ongoing disease monitoring in this species.