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The genetic relatedness of 672 penicillin-resistant isolates of Streptococcus pneumoniae (PRSP) recovered during national surveillance studies conducted in the United States during the periods of 1994–1995, 1997–1998, and 1999–2000 was determined by use of pulsed-field gel electrophoresis (PFGE). Overall, 104 different PFGE types were elucidated. For all study periods combined, the 12 most prevalent PFGE types included >75% of all isolates, and 5 types were closely related to widespread clones (Spain23F-1, France9V-3, Spain6B-2, Tennessee23F-4, and Taiwan19F-14). From 1994–1995 to 1999–2000, 3 major PFGE types (not closely related to 16 recognized clones) increased in prevalence. Multidrug resistance was identified among 96%–100% of the isolates in 9 of 12 predominant PFGE types. The prevalence of erythromycin resistance increased within 4 major PFGE types. These observations support the hypothesis that the dominant factor in the emergence of PRSP in the United States during the 1990s has been human-to-human spread of relatively few clonal groups that harbor resistance determinants to multiple classes of antibiotics.