Fluoroquinolones are not indicated for use for the treatment of pneumonia in children; however, non-levofloxacin-susceptible Streptococcus pneumoniae (NLSSP) has emerged in South Africa among children receiving treatment for multidrug-resistant tuberculosis. This study aimed to genotypically characterize NLSSP isolates. Invasive isolates were collected through active national laboratory-based surveillance for invasive pneumococcal disease (IPD) from 2000 through 2006 (n = 19,404). Carriage studies were conducted at two hospitals for patients with tuberculosis in two provinces. Phenotypic characterization was performed by determination of MICs and serotyping. Fluoroquinolone resistance mutations were identified, and clonality was investigated by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. Twelve non-levofloxacin-susceptible cases of IPD were identified, and all were in children <15 years of age. Ten isolates were serotype 19F and formed two clusters according to their PFGE profiles, antibiogram types, and fluoroquinolone resistance-conferring mutations. All nine carriage isolates from children in hospital A were NLSSP, serotype 19F, were indistinguishable by PFGE, and were related to invasive isolates in cluster 2. Of 26 child carriers in hospital B, 22 (85%) were colonized with NLSSP. The isolates were indistinguishable by PFGE, although they displayed two serotypes, serotypes 19F and 23F. The isolates were related to invasive isolates in cluster 1; however, higher levofloxacin MICs and different fluoroquinolone resistance mutations were suggestive of horizontal gene transfer. A serotype 23F carriage isolate displayed increased fitness compared with the fitness of an otherwise indistinguishable serotype 19F carriage isolate. These data suggest that a low-level non-levofloxacin-susceptible strain transformed into a highly resistant strain under antibiotic pressure and underwent capsular switching in order to have increased fitness.