Toxoplasma gondii infection is a common cause of morbidity and mortality in New World primate species. Clinical abnormalities associated with toxoplasmosis can be nonspecific, making it difficult to make a definitive antemortem diagnosis and initiate appropriate treatment. Toxoplasmosis in New World primates can have a rapid clinical course, which may lessen the diagnostic utility of antemortem tests. However, while there are a variety of T. gondii serum antibody tests and T. gondii polymerase chain reaction (PCR) assays available that are not species specific, these assays have not been comparatively applied to New World primate cases. Woolly monkeys (Lagothrix lagotricha), a species of New World primate, are highly susceptible to fatal toxoplasmosis. Archived serum samples from 15 living and deceased woolly monkeys housed at the Louisville Zoological Garden (Louisville, Kentucky) were tested for T. gondii antibodies by a commercially available latex agglutination kit, a commercially available indirect hemagglutination kit, and the modified agglutination test. In addition, aliquots of the sera were assayed for T. gondii DNA using a PCR assay. Both woolly monkeys that died of disseminated toxoplasmosis were positive in all four assays, indicating that each could be used to aid in the diagnosis of toxoplasmosis in this species. We suspect that these assays have applications to other species of New World primates.