Streptococcus pneumoniae serotype 6C, which was described in 2007, causes invasive disease in adults and children. We investigated the prevalence of 6C among pediatric isolates obtained from eight children's hospitals in the United States. S. pneumoniae isolates were identified from a prospective multicenter study (1993 to 2009). Fifty-seven serotype 6C isolates were identified by multiplex PCR and/or Quellung reaction. Five were isolated before 2000, and the prevalence increased over time (P < 0.000001). The median patient age was 2.1 years (range, 0.2 to 22.5 years). Clinical presentations included bacteremia (n = 24), meningitis (n = 7), pneumonia (n = 4), abscess/wound (n = 3), mastoiditis (n = 2), cellulitis (n = 2), peritonitis (n = 1), septic arthritis (n = 1), otitis media (n = 10), and sinusitis (n = 3). By broth microdilution, 43/44 invasive serotype 6C isolates were susceptible to penicillin (median MIC, 0.015 mug/ml; range, 0.008 to 2 mug/ml); all were susceptible to ceftriaxone (median MIC, 0.015 mug/ml; range, 0.008 to 1 mug/ml). By disk diffusion, 16/44 invasive isolates (36%) were nonsusceptible to erythromycin, 19 isolates (43%) were nonsusceptible to trimethoprim-sulfamethoxazole (TMP-SMX), and all isolates were clindamycin susceptible. Multilocus sequence typing (MLST) revealed 24 sequence types (STs); 9 were new to the MLST database. The two main clonal clusters (CCs) were ST473 and single-locus variants (SLVs) (n = 13) and ST1292 and SLVs (n = 23). ST1292 and SLVs had decreased antibiotic susceptibility. Serotype 6C causes disease in children in the United States. Emerging CC1292 expressed TMP-SMX resistance and decreased susceptibility to penicillin and ceftriaxone. Continued surveillance is needed to monitor changes in serotype prevalence and possible emergence of antibiotic resistance in pediatric pneumococcal disease.