Author summaryIncreasing knowlesi malaria cases and other zoonotic simian malaria caused by Plasmodium cynomolgi and Plasmodium inui in humans have added another dimension of complexity to malaria elimination. Unfortunately, the entomological perspective for this disease is scarce and understudied. Accordingly, we aimed to understand the bionomics and transmission efficiency of the simian malaria vectors, and to examine the genetic diversity and evolutionary pattern of their simian Plasmodium. Our study revealed that the Anopheles Leucosphyrus Group mosquitoes are highly potential competent vectors, and they represent a risk of human infection with zoonotic simian malaria in Southeast Asia. Plasmodium cynomolgi and P. inui were highly prevalent in mosquitoes collected from the present study, and they demonstrated close relationship with those from the vertebrate hosts, suggesting ongoing transmission between the vectors, macaques, and humans. With these constant microevolutionary processes, there are risks for both P. inui and P. cynomolgi to emerge and spread as a major public health problem, following the trend of P. knowlesi in Southeast Asia. BackgroundThe elimination of malaria in Southeast Asia has become more challenging as a result of rising knowlesi malaria cases. In addition, naturally occurring human infections with other zoonotic simian malaria caused by Plasmodium cynomolgi and Plasmodium inui adds another level of complexity in malaria elimination in this region. Unfortunately, data on vectors which are responsible for transmitting this zoonotic disease is very limited. Methodology/Principal findingsWe conducted longitudinal studies to investigate the entomological parameters of the simian malaria vectors and to examine the genetic diversity and evolutionary pattern of their simian Plasmodium. All the captured Anopheles mosquitoes were dissected to examine for the presence of oocysts, sporozoites and to determine the parous rate. Our study revealed that the Anopheles Leucosphyrus Group mosquitoes are highly potential competent vectors, as evidenced by their high rate of parity, survival and sporozoite infections in these mosquitoes. Thus, these mosquitoes represent a risk of human infection with zoonotic simian malaria in this region. Haplotype analysis on P. cynomolgi and P. inui, found in high prevalence in the Anopheles mosquitoes from this study, had shown close relationship between simian Plasmodium from the Anopheles mosquitoes with its vertebrate hosts. This directly signifies the ongoing transmission between the vector, macaques, and humans. Furthermore, population genetic analysis showed significant negative values which suggest that both Plasmodium species are undergoing population expansion. Conclusions/SignificanceWith constant microevolutionary processes, there are potential for both P. inui and P. cynomolgi to emerge and spread as a major public health problem, following the similar trend of P. knowlesi. Therefore, concerted vector studies in other parts of Southeast Asia are warranted to better comprehend the transmission dynamics of this zoonotic simian malaria which eventually would aid in the implementation of effective control measures in a rapidly changing environment.