The recent increase in the creation of trans-boundary protected areas and wildlife corridors between them lend importance to information on pathogen prevalence and transmission among wildlife species that will become connected. One such initiative is the Kavango Zambezi Transfrontier Conservation Area (KAZA/TFCA) of which Botswana’s Okavango Delta constitute a major contribution in terms of wildlife and ecosystems. Between 2008 and 2011, we collected serum samples from 14 lions (Panthera leo), four leopards (P. pardus), 19 spotted hyenas (Crocuta crocuta), and six cheetahs (Acinonyx jubatus) in the Okavango. Samples were tested for antibodies against: canine distemper virus (CDV), feline panleukopenia virus, enteric coronavirus, feline calicivirus, feline herpesvirus (FHV-1), and feline immunodeficiency virus (FIV). Evidence of exposure to all of these pathogens was found to varying degrees in at least one of the species sampled. High seroprevalence (> 90%) was only found for FHV-1 and FIV in lions. Only hyenas (26%, 5/19) were seropositive against CDV. Apart from one case, all individuals displayed physical conditions consistent with normal health for a minimum of 12 months following sampling. Our results emphasize the need for a comprehensive multi-species approach to disease monitoring and the development of coordinated management strategies for sub-populations likely to be connected in trans-boundary initiatives.