A prospective study was performed including all children younger than 18 years with the clinical diagnosis of invasive meningococcal disease (IMD) hospitalized at the University Hospital Sant Joan de Déu in Barcelona, Spain, from January 2001 to December 2006. During the study period, 168 meningococcal disease cases were reported. Microbiologic confirmation was obtained in 118 cases. Forty-six (38.9%) of 118 cases were only detected by polymerase chain reaction (PCR); 6 patients were culture positive and PCR negative (5%). Serogroup B predominated in the 6-year period with 83.1% of the strains. A significant decrease in serogroup C was observed in the last 3 years of the study (P=0.029), and less common serogroups, such as serogroup A and W135, emerged. Serogroup distribution of patient diagnoses only by real-time PCR showed a similar distribution: serogroup B, 85.7%; serogroup C, 7.1%; and nontypeable serogroups, 7.1%. In conclusion, real-time PCR is more rapid and sensitive than culture for diagnosis and serogrouping of IMD.