In Colombia, penicillin resistance of Streptococcus pneumoniae invasive isolates recovered from children less than 5 years old has increased from 10% in 1994 to 49.4% in 1999, suggesting the circulation of international resistant clones in the country. A total of 167 S. pneumoniae invasive isolates with diminished susceptibility to penicillin (DSP) were studied. The techniques used were pulsed-field gel electrophoresis (PFGE) and restriction fragment length polymorphism (RFLP) of penicillin-binding proteins (PBPs) genes (2B, 2X, and 1A). Forty-nine serotype 23F isolates were grouped in two clusters: 15 (31%) multiresistant isolates showed PFGE pattern A and PBP I profile, thus making them indistinguishable from Spain23F-1 clone, and 34 (69%) with PFGE pattern C, PBP II profile, and intermediate level resistance (ILR) to penicillin and TMP-SMX, features unique to a Colombian clone. Fifty-five serotype 14 isolates were assigned to PFGE B pattern, PBP III profile, having high-level resistance to penicillin, and TMP-SMX, similar to the France9V variant 14. This same pattern was present in five capsular type 9V isolates. Four serotype 14 isolates were assigned to PFGE pattern F, and appeared to be similar to Slovakia(14)-10 PFGE pattern, although they had different PBP profiles. Nine capsular type 6B and one 6A isolates belonged to PFGE pattern M, similar to Spain6B-2, although they showed different PBP profiles. The remaining 44 isolates, corresponding to serotypes 14, 6B, 19F, and 34, showed variable PFGE and PBP patterns. These results show that as many as two international clones may be circulating in Colombia as well as a unique, widely distributed 23F clone with ILR to penicillin. Additionally, some Colombian isolates capsular type 14 and 6B might be related to Slovakia(14)-10 Spain6B-2 clones, respectively.