In previous studies, it was shown that in peri-domestic and urban areas of Singapore, Peninsular Malaysia, and Thailand, the parasitaemia and the presence of Plasmodium infections in free-ranging macaques were low or zero, hypothetically due to the absence or limited presence of known competent vectors of malaria parasites [17, 25, 39]. A comparison with multiple studies using molecular methods to detect Plasmodium infections in macaques from other locations indicates that the prevalence of malaria and of the different species of Plasmodium vary according to the sampling site, even on a small island like Singapore [18–20, 22–26]. Comparing the Singaporean samples screened from this study, which were collected from 2007, with the Singaporean samples reported from 2011 [20], there was a decrease in the prevalence of P. knowlesi infection. The exact location from where the macaques were trapped in Singapore is not known for the current study and the differences in the prevalence of P. knowlesi between the two studies could be due to different sampling locations in Singapore. In addition, in a study conducted in Sabah, Malaysian Borneo, Fornace et al. [40] have shown spatial heterogeneity in P. knowlesi incidence depending on the land type. They found that the incidence of P. knowlesi varied between village, forests, and historical forest loss areas, which further indicates that environmental changes influence the transmission of P. knowlesi. More recently, Moyes and co-workers used a newly developed incidence–environment assessment model to investigate the distributions of reservoir hosts and Anopheline vectors of P. knowlesi in Southeast Asia and assessed their relationships with the environment [41]. Their analyses showed that the predicted distribution of both the long-tailed macaques and vectors of knowlesi malaria encompassed a wide range of habitats, most significantly in the northern part of their study area which included Myanmar, Thailand, Laos, Cambodia and Vietnam. These recent studies highlight that environmental variables possibly are the key driving force in determining the distribution of Plasmodium parasites.