We compared Streptococcus pneumoniae serotype 1 isolates causing disease among children in six geographic regions of the United States to determine genetic relatedness. Genomic fingerprints were determined by repetitive element polymorphism PCR (Rep-PCR). Multilocus sequence type characterization was performed on selected isolates. Four different genomic banding patterns were identified by Rep-PCR. One profile (clone 1) was predominant and matched sequence type 227.