We established the distribution of amino acid alterations in quinolone resistance-determining regions (QRDRs) of Streptococcus pneumoniae isolates in Japan and described the correlation of these alterations with serotypes determined by multilocus sequencing typing. Among 141 S. pneumoniae isolates, five levofloxacin-resistant isolates harbored mutations in both gyrA and parC and/or parE and were clonally unrelated. Among 136 levofloxacin-susceptible isolates, one isolate (MIC = 2 mg/l) had a first-step parC mutation at Asp78. Twenty isolates had Lys137Asp in parC and Ile460Val in parE and contained nine serotypes and eight clonal complexes (CCs), including all eight Colombia(23F)-26 (CC138) isolates. Eighty-one isolates had Ile460Val in parE alone and contained 14 serotypes and 16 CCs, including 36 of 37 Netherlands(3)-31 (CC180) isolates and all 22 Taiwan(19F)-14 (CC271) isolates. In contrast, seven of ten Taiwan(23F)-15 (CC242) isolates were wild-type. Although each QRDR genotype contained various serotypes and CCs, prevalent clones were mostly associated with a single QRDR genotype.