Three isolates of Streptococcus agalactiae, recovered from residents of the metropolitan area of Rio de Janeiro with significant bacteriuria, were found to be resistant to levofloxacin. Determination of the minimal inhibitory concentration (MIC) confirmed one isolate as intermediate and two as resistant to levofloxacin. No reduction in levofloxacin MIC was observed with reserpine, indicating that resistance was not caused by an efflux mechanism. Typical point mutations were observed in the quinolone resistance determinant region of gyrA and parC. Other point mutations in parC generated novel altered codons: Ser80→Pro in the intermediate resistance isolate, and Gly128→Asp in a resistant isolate. Through molecular modeling, it was possible to observe that these novel substitutions might not play a role in resistance, since these amino acids were not involved in the antibiotic binding site. Pulsed field gel electrophoresis profiles revealed a non-clonal trend among these isolates. This is the first report of genetic characterization of levofloxacin-resistant S. agalactiae strains in Brazil.