A real-time PCR assay with conventional microscopy by Giemsa-stained blood films was used. PCR was completed in an hour and identified the Plasmodium species in a single reaction. Blood was collected, and DNA was extracted. A genus-specific primer set corresponding to 18S ribosomal RNA was used to amplify target sequence. Fluorescence resonance energy technology hybridization probes were designed for P. falciparum over a region containing base pair mismatches allowed Plasmodium species differentiation. Microscopically positive patients (n = 60) were positive with real-time assay (100% sensitivity). 58 were single-species infections caused by P. falciparum; mixed infections (P. falciparum & P. vivax) were shown by real-time assay. Six out of 30 negative microscopy specimens were positive by real-time PCR (80% specificity). The discrepant results could be due to the subjective nature of microscopy and analytical objectivity of PCR, and high analytical sensitivity of real-time assay (1 parasite/microl) compared to microscopy (50 parasites/microl). Six patients were retested with ICT malaria test and 4 were positive showing that PCR results were correct. There was low correlation between parasitemia by microscopy and gene copy number for P. falciparum (r = 0.2; P = 0.05 [Spearman]).