<p><i>Toxoplasma gondii</i> (<i>T. gondii)</i> is a globally distributed protozoan parasite that can infect a wide range of warm-blooded animals, including humans. Nonhuman primates (NHPs) are particularly valuable for studying toxoplasmosis because of their physiological and immunological similarity to humans. In this study, we report the isolation and characterization of two <i>T. gondii</i> strains from captive NHPs that died suddenly in a zoo in Guangdong Province, China. The animals, a red-backed bearded saki (<i>Chiropotes chiropotes</i>), a New World monkey, and a mandrill (<i>Mandrillus sphinx</i>), an Old World monkey, were diagnosed with acute fatal toxoplasmosis on the basis of histopathology, immunohistochemistry, serology (MAT), and PCR. Parasite isolation was performed via a mouse bioassay, and the strains were genotyped using multilocus PCR–RFLP. The isolates were identified as ToxoDB #15 and ToxoDB #169, atypical genotypes that have been reported in Africa, Europe, and the Americas. Pathogenicity assessment in Swiss mice revealed that both TgMonkeyGd2 (ToxoDB #15) and TgMonkeyGd3 (ToxoDB #169) exhibited intermediate virulence, causing 100% and 80% mortality at 10⁴ tachyzoites, respectively, with lower doses allowing host survival and seroconversion. These findings challenge the traditional view that Old World monkeys (exemplified by the mandrill) are relatively resistant to toxoplasmosis and suggest a possible role of the wildlife trade in the transcontinental spread of exotic <i>T. gondii</i> genotypes, although this hypothesis requires further investigation with a larger number of cases. Enhanced surveillance and biosecurity measures are urgently needed in captive animal facilities to prevent outbreaks and protect both animal and public health.</p>