T he monkey parasite Plasmodium knowlesi was discovered to be a common cause of malaria in humans in 2004, initially from investigations in the Kapit division of Sarawak state, Malaysian Borneo (1). Humans acquire infection primarily from wild long-tailed (Macaca fascicularis) and pig-tailed (M. nemestrina) macaque reservoirs (2); Anopheles mosquitoes of the leucosphyrus group are vectors (3,4). P. knowlesi malaria has been described across Southeast Asia, but most clinical cases are still reported in Malaysian Borneo (3,5-8). In 2017 and 2018, a total of 7,745 cases were reported in Malaysia, 86.8% of which were detected in Malaysian Borneo (B. Singh, unpub. data) (9). P. knowlesi infections can be asymptomatic (10,11), and clinical cases exhibit a wide spectrum of disease ranging from mild symptoms to death (3). Population genetic surveys of P. knowlesi infections in humans across Malaysia have revealed 2 divergent subpopulations of the parasite in Malaysian Borneo that are associated with the 2 macaque species locally, suggesting 2 independent zoonoses (12,13). The cluster 1 type has been associated with longtailed macaques and the cluster 2 type with pig-tailed macaques (12). The existence of 2 sympatric subpopulations also has been confirmed by whole-genome sequencing (WGS) of P. knowlesi from patients in Malaysian Borneo (13,14). In peninsular Malaysia on the Asia mainland, all cases have been caused by another subpopulation, cluster 3, that has not been detected in Malaysian Borneo (13,15). Limited WGS (14,15) and microsatellite (13) genotyping of P. knowlesi isolates derived from human and only long-tailed macaque hosts from peninsular Malaysia showed allopatric divergence for this subpopulation cluster from those of Malaysian Borneo because of geographic separation by the South China Sea. Increasing numbers of P. knowlesi malaria cases detected might be due to increased zoonotic exposure along with a reduction of endemic malaria parasite species (16). With the recent identification of different zoonotic P. knowlesi genetic subpopulations, determining whether these populations vary in frequency over space and time is important. Interactions with