Malaria was eliminated from southern and southeastern Brazil over 50 years ago. However, an increasing number of autochthonous episodes attributed to Plasmodium vivax have recently been reported from the Atlantic Forest region of Rio de Janeiro state. As the P. vivax-like non-human primate malaria parasite Plasmodium simium is locally enzootic, we performed a molecular epidemiological investigation to determine whether zoonotic malaria transmission is occurring. We examined blood samples from patients presenting with malaria and from local howler monkeys by microscopy and PCR. Samples were included from individuals if they had a history of travel to or resided in areas within the Rio de Janeiro Atlantic Forest, but not if they had malaria prophylaxis, or had travelled to known malaria endemic areas in the preceding year. Additionally, we developed a molecular assay based on sequencing of the parasite mitochondrial genome to distinguish between P. vivax and P. simium, and applied this assay to 33 cases from outbreaks that occurred in 2015, and 2016. A total of 49 autochthonous malaria cases were reported in 2015–16. Most patients were male, with a mean age of 44 years (SD 14·6), and 82% lived in urban areas of Rio de Janeiro state and had visited the Atlantic Forest for leisure or work-related activities. 33 cases were used for mitochondrial DNA sequencing. The assay was successfully performed for 28 samples, and all were shown to be P. simium, indicative of zoonotic transmission of this species to human beings in this region. Sequencing of the whole mitochondrial genome of three of these cases showed that P. simium is most closely related to P. vivax parasites from South America.