The rapid increase of penicillin-resistant Streptococcus pneumoniae isolates could be a consequence of the spread of clones or due to the antimicrobial selective pressure. The genetic relatedness of 190 invasive isolates S. pneumoniae with reduced susceptibility to penicillin recovered from Colombian children less than 5 years old during a surveillance study from 2000 to 2003 was determined by the use of pulsed-field electrophoresis (PFGE). Overall, 42 different PFGE patterns were identified, but 4 of them included 76% of all isolates. They were related with international clones 1-Spain23F, 2-Spain6B, 3-Spain9V and 26-Colombia23F. Our results indicated that the dissemination of penicillin-resistant S. pneumoniae was the result of the spread of international clones, specially, the 3-Spain9V clone.