Since the 1970s, no information on simian malaria has been documented in Taiwan, an area that is free from human malaria. To update the prevalence of simian malaria, a molecular-based survey was performed. Blood samples from 286 Formosan macaques (Macaca cyclopis) were tested for Plasmodium species by microscopy and nested polymerase chain reaction. Furthermore, the field isolates were characterized by sequencing the 42-kDa fragment of the merozoite surface protein 1 (MSP-1₄₂). Of the 286 blood samples analyzed, 7 (2.4%) were positive by microscopy and nested PCR. All malaria-infected Formosan macaques were those collected from southern Taiwan, whereas no evidence of malarial parasites was observed among monkeys from eastern and northern Taiwan. Molecular and phylogenetic analyses based on the asexual stage small subunit ribosomal RNA (SSU rRNA) gene clearly identified these samples as a single infection with Plasmodium inui. Furthermore, phylogenetic analysis of the MSP-1₄₂ gene showed that the 7 field isolates were closely related to P. inui strains Taiwan I and II, which were obtained from Formosan macaques in 1963. These findings indicate that P. inui is the only cause of simian malaria in Taiwan, has been circulating in Formosan macaques at least for 46 yr, and has a geographic preference for southern Taiwan.