Despite recent success in reducing the regional incidence of Plasmodium falciparum malaria, cases of zoonotic malaria are on the rise in Southeast Asia. The Cambodian National Malaria Surveillance Program has previously relied on rapid diagnostic tests and blood smear microscopy with confirmatory polymerase chain reaction (PCR) testing in a subset of cases to further distinguish P. falciparum, P. malariae, P. ovale, and P. vivax species. Here, metagenomic next-generation sequencing identified P. knowlesi mono-infection in six Cambodian patients initially diagnosed with P. malariae by blood smear microscopy in February-May 2020. These findings of recent human infections with P. knowlesi in Cambodia led to the incorporation of P. knowlesi-specific PCR diagnostics to national malaria surveillance efforts.