Background: Microscopy remains the mainstay method for malaria diagnosis worldwide, although species misidentifications have been detected in practices due to various limitations, such as hypnozoites detection and lower parasitemia in asymptomatic malaria. Polymerase Chain Reaction (PCR) is a molecular diagnostic method with high accuracy in detecting species of organisms. This study was aimed to evaluate the diagnostic performance of microscopy compared to nested PCR in detecting malaria parasites. Methods: A cross-sectional study was conducted with previous data on malaria assessment in East Nusa Tenggara. More than 500 asymptomatic respondents were included by the systematic random sampling method from 5 sub-districts area in the region based on API. Microscopic assessment by thick and thin blood smears was made following protocols from the Ministry of Health, while DNA isolation was done using 200 µl fresh blood sample and nested PCR amplification protocol with specific primers of the malaria parasites species Plasmodium sp. Results: A total of 555 specimens were collected, and 1.6% (9/555) of those were microscopy-positive and 32.6% (181/555) were detected positive by nested PCR. Of microscopy-positive samples, 33.3% (3/9) were P. falciparum and 66.7% (6/9) were P. vivax, whereas among PCR-positive samples, 31.5% (57/181) were P. falciparum, 52.5% (95/181) were P. vivax, and 16.0% (29/181) were mixed infection of both species. From this study, microscopy was found to had a slight measure of agreement (κ = 0.055) compared to nested PCR. Conclusion: In lower parasitemia and asymptomatic malaria, the microscopic assessment may not be sensitive. Thus, this increases the need of using PCR assessment to confirm the identification of malaria parasites.