ABSTRACT.- The black-tufted marmoset (Callithrix penicillata), commonly found in urban areas of Central Brazil, is vulnerable to pathogen spillover from domestic animals and humans. Here, we report an outbreak of natural canine distemper virus (CDV) infection among urbanized free-ranging black-tufted marmosets. Five fatalities occurred in marmosets living in a neighborhood with unvaccinated dogs. Clinically, affected marmosets had lethargy, ataxia, mucocutaneous ulcerations, and crusting lesions. Postmortem findings included epithelial erosions, interstitial pneumonia, bronchopneumonia, and suppurative myocarditis, frequently associated with secondary bacterial infections. Immunohistochemistry (IHC) confirmed the presence of CDV antigen in multiple organs, and secondary bacterial infections were common, involving species, such as Bordetella, Haemophilus, and Streptococcus. Transmission electron microscopy (TEM) demonstrated paramyxovirus-like inclusion bodies and metagenomic sequencing identified a novel CDV variant. Phylogenetic analyses placed this strain within the Europe 1/South America 1 lineage, closely related to domestic dog-derived strains from the region. Comparative H gene analysis uncovered unique R519I substitutions in the CDV marmoset variant, suggesting potential for cross-species adaptation. This study provides evidence that CDV can naturally infect free-living New World primates, with possible implications for animal health, conservation, and interspecies transmission. These findings highlight the vulnerability of urban wildlife to CDV spillover from domestic dogs and emphasize the importance of monitoring pathogen transmission at the human-animal interface from a One Health perspective. Copyright © 2025 Tais Meziara Wilson et al. Transboundary and Emerging Diseases published by John Wiley & Sons Ltd.