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 byPlasmodium cynomolgiandPlasmodium inuiadds 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 simianPlasmodium. All the capturedAnophelesmosquitoes were dissected to examine for the presence of oocysts, sporozoites and to determine the parous rate. Our study revealed that theAnophelesLeucosphyrus 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 onP.cynomolgiandP.inui, found in high prevalence in theAnophelesmosquitoes from this study, had shown close relationship between simianPlasmodiumfrom theAnopheles 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 bothPlasmodiumspecies are undergoing population expansion.Conclusions/SignificanceWith constant microevolutionary processes, there are potential for bothP.inuiandP.cynomolgito emerge and spread as a major public health problem, following the similar trend ofP.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.