PCR-based assays are the most sensitive and specific methods to detect malaria parasites. This study compared the diagnostic accuracy of three PCR-based assays that do not only differ in their sequence target, but also in the number of copies of their target region, for the detection of Plasmodium falciparum in 401 individuals living in a malaria-endemic area in Nigeria. Compared to a composite reference generated from results of all the 3 PCR assays, the stevor gene amplification had a sensitivity of 100% (Kappa = 1; 95% CI = 1.000-1.000), 83% (Kappa = 0.718; 95% CI = 0.648-0.788) by SSUrRNA gene PCR and 71% (Kappa = 0.552; 95% CI = 0.478-0.627) by the msa-2 gene amplification. Results from this study indicate that the stevor gene amplification is the most sensitive technique for the detection of P. falciparum. This assay may be an important reference standard, especially when a confirmatory technique with high sensitivity and specificity is needed for ruling out P. falciparum infection.