Background Xenotransplantation is a potential answer to the current organ shortage, but the risk of infections related to overimmunosuppression is an important parameter that may predict the recipient's long-term survival. Cytomegalovirus (CMV) in xenotransplanted and immunosuppressed primates is a well-known cause of disease particularly affecting the gastrointestinal (GI) tract and a zoonotic concern. Methods Post-mortem sera and tissues from 45 immunosuppressed and xenografted Macaca fascicularis were evaluated for CMV using antisera specific for the immediate early 1 (IE1), anti-RhCMV, and QPCR for virus. Results Serological analysis showed 100% positivity for the presence of CMV antibodies following the application of a specific test designed for RhCMV. Five of 45 primates showed typical lesions of CMV infection in the GI tract, including neutrophilic enteritis and inclusion bodies. Molecular analysis confirmed the presence of recipient's CMV in the tissues with CMV histopathology. Porcine CMV from the donor animals was not found in any of the CMV-specific IHC-positive recipients. Conclusion The presence of active CMV infection in animals intended for xenograft experiments can lead to severe gastrointestinal lesions that could impact the overall aims of the study. In such cases, the animals should be investigated using appropriate (non-human primate-specific) diagnostic tools prior to use and treated aggressively with state-of-the-art antiviral therapy.