Neotropical primates are extremely sensitive to Toxoplasma gondii infection, with a high lethality rate. Infection and disease development occur most frequently through contact and ingestion of oocysts shed by felines in the soil. The aim of this study was to assess the frequency of anti-T. gondii antibodies in serum samples from 77 primates treated at the Center for Research and Medicine of Wild Animals (CEMPAS) at the School of Veterinary Medicine and Animal Science, UNESP, Botucatu, SP, Brazil, as well as free-living primates from Vitória do Xingu, PA, in brazilian Amazonia, using the indirect fluorescent antibody test (IFAT) and modified agglutination test (MAT). Considering the MAT results, frequency of anti-T. gondii antibodies was higher in Alouatta guariba (14/24; 58.33%; 95% CI: 38.66-75.60), followed by Sapajus nigritus (3/22; 13.64%; 95% CI: 4.95-33.59) and Alouatta belzebul (2/30; 6.67%; 95% CI: 2.04-21.42). Cebus olivaceous had only one animal evaluated, which reacted to MAT. Frequency of reactive animals was higher in those from captivity (33.33%; 95% CI: 20.21-49.79) in contrast to those in the wild (19.51%; 95% CI: 10.30-34.11), without significance (P > 0.20). Regarding IFAT, three samples were positive, revealing a low Kappa association index (0.21) compared to MAD, with a positive agreement of 26.09%. Based on the results, the use of MAD are recommended for evaluating serum samples from Neotropical primates due to its ease of execution and absence of the need for species-specific reagents or specialized equipment.