Background:ZoonoticP. knowlesiandP. cynomolgisymptomatic and asymptomatic infections occur across endemic areas of Southeast Asia. Most infections are low-parasitemia, with an unknown proportion below routine microscopy detection thresholds. Molecular surveillance tools optimizing the limit of detection (LOD) would allow more accurate estimates of zoonotic malaria prevalence.Methods:An established ultra-sensitivePlasmodiumgenus quantitative-PCR (qPCR) assay targeting the 18S rRNA gene underwent LOD evaluation with and without reverse transcription (RT) forP. knowlesi, P. cynomolgiandP. vivaxusing total nucleic acid preserved (DNA/RNA ShieldTM) isolates and archived dried blood spots (DBS). LODs for selectedP. knowlesi-specific assays, and referenceP. vivax- andP. cynomolgi-specific assays were determined with RT. Assay specificities were assessed using clinical malaria samples and malaria-negative controls.Results:The use of reverse transcription improvedPlasmodiumspecies detection by up to 10,000-fold (Plasmodiumgenus), 2759-fold (P. knowlesi), 1000-fold (P. vivax) and 10-fold (P. cynomolgi). The median LOD with RT for the Kamau et al.Plasmodiumgenus RT-qPCR assay was ≤0.0002 parasites/µL forP. knowlesiand 0.002 parasites/µL for bothP. cynomolgiandP. vivax. The LODs with RT forP. knowlesi-specific PCRs were: Imwong et al. 18S rRNA (0.0007 parasites/µL); Divis et al. real-time 18S rRNA (0.0002 parasites/µL); Lubis et al. hemi-nested SICAvar (1.1 parasites/µL) and Lee et al. nested 18S rRNA (11 parasites/µL). The LOD forP. vivax- andP. cynomolgi-specific assays with RT were 0.02 and 0.20 parasites/µL respectively. For DBSP. knowlesisamples the median LOD for thePlasmodiumgenus qPCR with RT was 0.08, and without RT was 19.89 parasites/uL (249-fold change); no LOD improvement was demonstrated in DBS archived beyond 6 years. ThePlasmodiumgenus andP. knowlesi-assays were 100% specific forPlasmodiumspecies andP. knowlesidetection, respectively, from 190 clinical infections and 48 healthy controls. ReferenceP. vivax-specific primers demonstrated known cross-reactivity withP. cynomolgi.Conclusion:Our findings support the use of an 18S rRNAPlasmodiumgenus qPCR and species-specific nested PCR protocol with RT for highly-sensitive surveillance of zoonotic and humanPlasmodiumspecies infections.