We describe a tuberculosis outbreak caused by Mycobacterium bovis in a conditioned colony of rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques. Animals in five rooms were exposed, but most (16/27) infections were confined to the room that housed a mixed population of cynomolgus and rhesus macaques. In this room, rhesus (8/8) and cynomolgus (10/11) macaques naturally exposed to M. bovis were infected at nearly identical rates (Fisher exact test, 2-tailed P = 1). The clinical signs of disease and pathologic lesions in infected macaques, however, were moderately different between the two species. Rhesus macaques were more likely (5/8) to exhibit clinical signs of persistent coughing and inappetance, and had more severe pulmonary lesions. By contrast, clinical signs of disease were seen in only 1 of 19 cynomolgus macaques, and overall, the pulmonary lesions were often focal and less severe, although some still had severe involvement of the lungs similar to that seen in rhesus macaques. These differences should be taken into consideration when developing or evaluating a tuberculosis-screening program. On the basis of observations made during this outbreak, we recommend that alternative screening methods such as the PRIMAGAM test and the ESAT-6 ELISA, be incorporated into the screening program to aid in the identification of infected animals.