Naturally acquired human infections with Plasmodium knowlesi are endemic to Southeast Asia. To determine the prevalence of P. knowlesi malaria in malaria-endemic areas of Thailand, we analyzed genetic characteristics of P. knowlesi circulating among naturally infected macaques and humans. This study in 2008-2009 and retrospective analysis of malaria species in human blood samples obtained in 1996 from 1 of these areas showed that P. knowlesi accounted for 0.67% and 0.48% of human malaria cases, respectively, indicating that this simian parasite is not a newly emergent human pathogen in Thailand. Sequence analysis of the complete merozoite surface protein 1 gene of P. knowlesi from 10 human and 5 macaque blood samples showed considerable genetic diversity among isolates. The sequence from 1 patient was identical with that from a pig-tailed macaque living in the same locality, suggesting cross-transmission of P. knowlesi from naturally infected macaques to humans.