Background: Plasmodium knowlesi and Plasmodium cynomolgi are two malaria parasites naturally transmissible between humans and wild macaque through mosquito vectors, while Plasmodium inui can be experimentally transmitted from macaques to humans. One of their major natural hosts, the long-tailed macaque (Macaca fascicularis), is host to two other species of Plasmodium (Plasmodium fieldi and Plasmodium coatneyi) and is widely distributed in Southeast Asia. This study aims to determine the distribution of wild macaques infected with malarial parasites by examining samples derived from seven populations in five countries across Southeast Asia.Methods: Plasmodium knowlesi, P. cynomolgi, P. coatneyi, P. inui and P. fieldi, were detected using nested PCR assays in DNA samples from 276 wild-caught long-tailed macaques. These samples had been derived from macaques captured at seven locations, two each in the Philippines (n = 68) and Indonesia (n = 70), and one each in Cambodia (n = 54), Singapore (n = 40) and Laos (n = 44). The results were compared with previous studies of malaria parasites in longtailed macaques from other locations in Southeast Asia. Fisher exact test and Chi square test were used to examine the geographic bias of the distribution of Plasmodium species in the macaque populations. Results: Out of 276 samples tested, 177 were Plasmodium-positive, with P. cynomolgi being the most common and widely distributed among all long-tailed macaque populations (53.3 %) and occurring in all populations examined, followed by P. coatneyi (20.4 %), P. inui (12.3 %), P. fieldi (3.4 %) and P. knowlesi (0.4 %). One P. knowlesi infection was detected in a macaque from Laos, representing the first documented case of P. knowlesi in wildlife in Laos. Chi square test showed three of the five parasites (P. knowlesi, P. coatneyi, P. cynomolgi) with significant bias in prevalence towards macaques from Malaysian Borneo, Cambodia, and Southern Sumatra, respectively. Conclusions: The prevalence of malaria parasites, including those that are transmissible to humans, varied among all sampled regional populations of long-tailed macaques in Southeast Asia. The new discovery of P. knowlesi infection in Laos, and the high prevalence of P. cynomolgi infections in wild macaques in general, indicate the strong need of public advocacy in related countries.