Six drug-resistant Streptococcus pneumoniae clones were previously identified from day care centers in Portugal, primarily on the basis of common pulsed-field gel electrophoresis (PFGE) patterns. These clones were susceptible to penicillin or had only very low-level resistance to it (most MICs, < or =0.25 microg/mL) and accounted for a large proportion (35%) of all drug-resistant pneumococci colonizing the nasopharynx of healthy children attending day care. Five of the 6 clones were identified among pneumococcal clinical isolates collected in other countries. In this study, we applied multilocus sequence typing (MLST) to describe the genetic background of these clones. MLST confirmed previous findings obtained by PFGE and allowed for the extension of the international clonal relationships by showing that each of the 6 clones was internationally disseminated and was able to cause pneumococcal disease.