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To detect relatedness among 68 (0.5%) of 13 795 US clinical isolates of Streptococcus pneumoniae from the TRUST 3 (1998–1999) and TRUST 4 (1999–2000) surveillance studies that were resistant to levofloxacin (MIC ≥ 8 mg/L).All levofloxacin-resistant isolates were analysed by broth microdilution reference method for susceptibility to four fluoroquinolones, DNA sequencing of quinolone resistance determining region (QRDR) of topoisomerase IV and DNA gyrase genes, serotyping, and pulsed-field gel electrophoresis (PFGE).All levofloxacin-resistant isolates were ciprofloxacin resistant (MIC ≥ 4 mg/L, FDA breakpoint) and non-susceptible to gatifloxacin (MIC ≥ 2 mg/L); 62 were non-susceptible to moxifloxacin (MIC ≥ 2 mg/L). Resistant isolates were in 48 (20%) of 238 institutions in 29 states. Three institutions had levofloxacin-resistant isolates in both surveillance studies. Among the resistant isolates were 17 serotypes and 48 different PFGE patterns. Fourteen isolates had PFGE patterns closely related to the Spain23F-1 clone; one strain had a PFGE pattern closely related to the French9V-3 clone. All levofloxacin-resistant isolates had two or more mutations within the QRDR of parC, parE, gyrA and gyrB.US levofloxacin-resistant S. pneumoniae isolates were rare and most were unrelated with minimal clonal spread, and were associated with multiple QRDR mutations with extensive cross-resistance noted among fluoroquinolones.