Although Mycobacterium simiae was identified and classified more than three decades ago, only a few cases are mentioned in the current literature. After experimental simian immunodeficiency virus infection, a 9-year-old female rhesus monkey (Macaca mulatta) developed progressive immunosuppression and gastrointestinal disease very similar to the clinical and pathomorphologic features of Johne's disease, which is caused by M. paratuberculosis. Acid-fast-positive bacteria reacted immunohistochemically with antibodies against M. paratuberculosis and M. bovis but were not useful for differentiation because of a high degree of cross-reactivity. In contrast to immunohistochemistry and histopathology, biochemical methods and cycle sequencing analysis of the 16S ribosomal RNA identified M. simiae as the disease-causing pathogen. This case demonstrates the importance of molecular biological methods for the diagnosis of M. simiae infection in monkeys.