Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis) infection, is a multi-host disease which negatively affects the wildlife industry, with adverse consequences for conservation, ecotourism, and game/wildlife sales. Although interspecies transmission has been reported between some wildlife hosts, the risk of spread in complex ecosystems is largely unknown. As a controlled disease, tools for accurate detection of M. bovis infection is crucial for effective surveillance and management, especially in wildlife populations. There are however, limited species-specific diagnostic tests available for wildlife. Hippopotamuses are rarely tested for M. bovis infection and infection has not previously been confirmed in these species. In this study, blood and tissue samples collected from common hippopotamus (Hippopotamus amphibius) residing in a bTB endemic area, the Greater Kruger Protected area (GKPA), were retrospectively screened to determine whether there was evidence for interspecies transmission of M. bovis, and to identify tools for M. bovis detection in this species. Using the multi-species DPP® VetTB serological assay, a bTB seroprevelance of 8% was found in hippopotamus from GKPA. In addition, the first confirmed case of M. bovis infection in a free-ranging common hippopotamus is reported, based on the isolation in mycobacterial culture, genetic speciation, and detection of DNA in tissue samples. Importantly, the M. bovis spoligotype (SB0121) isolated from this common hippopotamus is shared with other M. bovis-infected hosts in GKPA, suggesting interspecies transmission. These results support the hypothesis that M. bovis infection may be under recognized in hippopotamus. Further investigation is needed to determine the risk of interspecies transmission of M. bovis to common hippopotamus in bTB endemic ecosystems and evaluate serological and other diagnostic tools in this species.