Lion (Panthera leo) populations, classified as vulnerable under the International Union for Conservation of Nature red list of threatened species, are facing a variety of threats, including tuberculosis (TB) caused by Mycobacterium bovis. The lack of knowledge on pathogenesis and diagnosis of TB, the prolonged course of the disease, the existence of subclinical infection, and nonspecific clinical signs hamper management of TB in both free-ranging and captive lion populations. Early and accurate antemortem diagnosis of M. bovis infections is important for disease management. In this study, we investigate the suitability of the single intradermal cervical test (SICT), developed with free-ranging Kruger National Park (KNP) lions exposed to M. bovis, for use in other lion populations. Using the recommended interpretation, the specificity of the SICT was low in disease-free captive lions, leading to false-positive diagnoses in 54% of individuals in the present study. Alternative interpretations of the tuberculin skin test are proposed that significantly reduce false-positive diagnosis in the sampled captive lions without significantly affecting diagnoses in the KNP lions; these changes may facilitate screening for M. bovis infection regardless of the exposure status of the lion population being investigated.