A newly described pneumococcal serotype (6C) is indistinguishable from serotype 6A when using the conventional Quellung serotyping method. Serotype 6A isolates were screened by polymerase chain reaction (PCR) for the wciN region of the capsular locus. This study detected serotype 6C among invasive pneumococcal disease (IPD) isolates from national laboratory-based surveillance (2005-2006) in South Africa. No serotype 6C isolates were identified among 23 serotype 6A cases from children enrolled in a 9-valent pneumococcal conjugate vaccine trial (1998-2005). Of 8167 IPD cases reported nationally, viable isolates were available for serotyping in 87% of cases (n=7080). Quellung serotyping identified 608 serotype 6A isolates, of which 606 were further tested for serotype 6C. PCR confirmed serotype 6C in 5% (30/606) of the isolates tested. Serotype 6C isolates were: less likely than 6A to cause disease in children compared with adults (6/30 (20%) vs. 311/550 (57%); P<0.001); more likely to cause laboratory-confirmed meningitis (15/30 (50%) vs. 167/578 (29%); P=0.01); and more likely to demonstrate susceptibility to penicillin (non-susceptibility 0/30 vs. 129/578 (22%); P=0.004). No association with gender, human immunodeficiency virus (HIV) co-infection or case fatality rate was observed. Although serotype 6C prevalence was low, its epidemiology may differ from the other serogroup 6 pneumococci. Our data from the vaccine efficacy trial suggest that cross-protection of the conjugate vaccine is against true serotype 6A strains.