The aims of this study were to evaluate the incidence of invasive pneumococcal disease (IPD) in Italian children and perform serotyping by PCR-based assays directly on clinical samples. A 1-year paediatric (0-14 years) population-based surveillance study was designed to evaluate the incidence of IPD in the province of Florence, Italy, by cultural and molecular methods. Among 92 children (80 with pneumonia, 8 with meningitis/sepsis, 4 with arthritis), 4 cases of IPD were diagnosed both by culture and real-time PCR and 18 cases exclusively by molecular methods. The sensitivity of molecular methods was significantly higher than that of cultural methods (Cohen's kappa 0.41; McNemar P=0.000008). The incidence of IPD in children below 2 years of age was 11.5/100,000 and 51.8/100,000 by cultural and molecular methods, respectively. Pneumococcal serotyping by multiplex sequential PCR was obtained in 19/22 samples. Real-time PCR and multiplex sequential PCR can be used directly on biological samples, improving the ability to diagnose IPD. The incidence of IPD appears 5-10 times higher by PCR than by cultural methods.