Recent studies using an internal transcribed spacer (ITS)-based real-time polymerase chain reaction (PCR) for the detection of Schistosoma DNA in urine samples has shown high sensitivity and specificity when performed on controls and known microscopy-positive samples. In this study, using 730 urine samples collected from children in five primary schools from different communities in the Greater Accra region of Ghana, specific detection of Schistosoma DNA showed excellent sensitivity of 100% and 85.2% in urines with > 50 eggs/10 mL urine and ≤ 50 eggs/10 mL of urine, respectively. Additionally, Schistosoma-specific DNA was amplified in 102 of 673 samples in which Schistosoma eggs could not be detected with microscopy. Taking microscopy and/or PCR-positive samples as true positives, the negative predictive value calculated was 94.6-100% for each school sampled as compared with 54.3-95.7% using microscopy. This ITS-based real-time PCR proves to be a powerful tool in epidemiological surveys of schistosomiasis providing more precise and sensitive results than microscopy.