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Background: Zoonotic P. knowlesi and P. cynomolgi symptomatic 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-sensitive Plasmodium genus quantitative-PCR (qPCR) assay targeting the 18S rRNA gene underwent LOD evaluation with and without reverse transcription (RT) for P. knowlesi , P. cynomolgi and P. vivax using total nucleic acid preserved (DNA/RNA Shield ™ ) isolates and archived dried blood spots (DBS). LODs for selected P. knowlesi- specific assays, and reference P. vivax- and P. 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 improved Plasmodium species detection by up to 10,000-fold ( Plasmodium genus), 2759-fold ( P. knowlesi ), 1000-fold ( P. vivax ) and 10-fold ( P. cynomolgi ). The median LOD with RT for the Kamau et al. Plasmodium genus RT-qPCR assay was ≤0.0002 parasites/μL for P. knowlesi and 0.002 parasites/μL for both P. cynomolgi and P. vivax . The LODs with RT for P. 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 for P. vivax- and P. cynomolgi- specific assays with RT were 0.02 and 0.20 parasites/μL respectively. For DBS P. knowlesi samples the median LOD for the Plasmodium genus 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. The Plasmodium genus and P. knowlesi -assays were 100% specific for Plasmodium species and P. knowlesi detection, respectively, from 190 clinical infections and 48 healthy controls. Reference P. vivax- specific primers demonstrated known cross-reactivity with P. cynomolgi ., Conclusion: Our findings support the use of an 18S rRNA Plasmodium genus qPCR and species-specific nested PCR protocol with RT for highly-sensitive surveillance of zoonotic and human Plasmodium species infections.