The states that make up the Legal Amazon Region, which include the state of Maranhão, account for 99% of registered cases of human malaria in Brazil. It is also believed that transmission of malaria from nonhuman primates (NHP) to humans occurs in this region, because of current reports of seroepidemiological results from samples from humans and NHP coexisting in the same areas. This study aimed to make morphological, serological and molecular diagnoses of Plasmodium spp. in neotropical primates on the island of São Luís, state of Maranhão, Brazil. The diagnostic techniques used were optical microscopy, the polymerase chain reaction (PCR) and the indirect immunofluorescence assay (IFA). From June 2009 to April 2010, 70 NHP were sampled: 50 at the Wild Animal Screening Center (CETAS), located in the municipality of São Luís and 20 free-living individuals that were caught in a private reserve located in the municipality of São Jose de Ribamar, state of Maranhão. Under an optical microscope, 140 slides (two from each animal) were evaluated and five animals (7.1%) were found to be positive. IFA did not detect anti-Plasmodium spp. From PCR on the 70 animals sampled, amplified Plasmodium spp. products were observed in 13 samples, of which eight (61.5%) were from free-living animals and five (38.5%) were from animals at CETAS.