The purpose of this study was to investigate the genetic characteristics of fluoroquinolone-nonsusceptible Streptococcus pneumoniae clinical isolates in China. A total of 377 S. pneumoniae clinical isolates, including 307 pediatric strains and 70 adult strains, were collected from eight centers in China. The minimal inhibitory concentrations (MICs) of 10 antimicrobial agents were determined by agar dilution. Multilocus sequence typing (MLST), serotyping, and quinolone resistance-determining region (QRDR) variations were conducted in levofloxacin-nonsusceptible isolates by polymerase chain reaction (PCR)-based methods. Seven levofloxacin-nonsusceptible isolates were found, with an overall resistance rate of 1.9 % (7/377) and 8.6 % (6/70) in adults. Sequence analyses of parC, gyrA, and parE QRDRs in levofloxacin-resistant isolates demonstrated mutations in dual target sites at the hot spots. These seven strains represented multiple clones: two strains were serotype 19F (Taiwan(19F)-14) and MLST clonal complex (CC) 271/320, two were typed as 23F (Spain(23F)-1) and CC81, two were determined as serotype 20B and a new sequence type of ST6935, and one non-serotypeable pediatric strain belonged to a new sequence type of ST6946. Two serotype 19F strains possessed a variety of characteristic alterations of viridans group streptococci in gyrA (Ser114Gly) or parC (Ser52Gly, Asn91Asp). Fluoroquinolone-nonsusceptible S. pneumoniae isolates showed a substantial degree of genetic diversity and belonged to pre-existing epidemic clones together with native clones. S. pneumoniae strains with serotype 20B was recovered for the first time to be associated with levofloxacin resistance in China.