BackgroundMalaria is a mosquito-borne disease that is one of the most serious public health issues globally and a leading cause of mortality in many developing countries worldwide. Knowing the prevalence of both symptomatic and asymptomatic malaria on a subnational scale allows for the estimation of the burden of parasitaemia present in the transmission system, enabling targeting and tailoring of resources towards greater impact and better use of available capacity. This study aimed to determine the PCR-based point prevalence of malaria infection, by parasite species, among three high-risk populations in Mondulkiri province, Cambodia: forest rangers, forest dwellers, and forest goers.MethodsA cross-sectional survey was performed during the transmission season in November and December 2021. Blood samples collected on filter paper from participants (n = 1301) from all target groups were screened forPlasmodiumspp using PCR.ResultsMalaria prevalence among all study participants was 6.7% for anyPlasmodiumspecies. Malaria prevalence in the forest ranger group was 8.1%, was 6.8% in forest goers, and 6.4% in forest dwellers; all infections were asymptomatic.Plasmodium vivaxwas detected in all participant groups, while the fewPlasmodium falciparuminfections were found in goers and dwellers. 81% of all infections were due toP. vivax, 9% were due toP. falciparum, 3% due toPlasmodium cynomolgi, and the rest (7%) remained undefined. Gender was associated with malaria infection prevalence, with male participants having higher odds of malaria infection than female participants (OR = 1.69, 95% CI 1.08–2.64). Passively collected malaria incidence data from the Cambodian government were also investigated. Health facility-reported malaria cases, based on rapid diagnostic tests, for the period Jan-Dec 2021 were 521Plasmodium vivax(0.89% prevalence),34P. falciparum(0.06%) and fourP. falciparum+ mixed (0.01%)—a total of 559 cases (0.95%) for all of Mondulkiri.ConclusionThis reservoir of asymptomatic parasitaemia may be perpetuating low levels of transmission, and thus, new strategies are required to realize the goal of eliminating malaria in Cambodia by 2025.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12936-023-04630-2.