A total of 773 pneumococcal isolates were collected from a nationwide surveillance of invasive pneumococcal diseases during 1999-2003 prior to the implementation of the 7-valent conjugate vaccine (PCV7) in Italy. The isolates included vaccine serotypes (VS, 393 isolates), vaccine-related serotypes (VRS, 93), and nonvaccine serotypes (NVS, 279). The ten most prevalent serotypes were: 14 (16.4%), 3 (8.4%), 23F (8%), 19F (7.4%), 4 (5.9%), 7F (5.8%), 9V (5.3%), 6B (4.9%), 19A (4.7%), and 1 (3.7%). VRS or NVS isolates showed a lower rate of penicillin or drug resistance than VS. Representative isolates of the major VS, VRS, and NVS were genotyped by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The isolates examined were found to belong to 18 international clones and to eight newly described clones. VS isolates sharing clonal groups with VRS or NVS were also detected. Evidence of a past history of capsular switching events was observed in five clones.