The aim of this study was to determine the prevalence of plasmid-mediated qnr genes among 61 consecutive nonrepetitive clinical strains of E. coli isolated from dogs (n 41) and cats (n 20) with urinary tract infections at the Faculty of Veterinary Medicine in Lisbon, Portugal, from 2004 to 2006, and to subsequently characterize positive isolates. Thirty-four percent and 26% of the strains were resistant to nalidixic acid and ciprofloxacin, respectively. Two were CMY-2 producers, and only one was an ESBL-producing strain. The qnrA and qnrS genes were not found. The qnrB gene was detected in one (1.6%) of 61 E. coli isolates, which was identified as qnrB2. The qnrB2-positive strain, FMV5825, isolated from a 15-year-old female dog with chronic cystitis, was resistant to different antibiotics including ampicillin, cephalosporins (except 7--methoxy-cephalosporins), aztreonam, piperacillin, ticarcillin, trimethoprim-sulfamethoxazole, aminoglycosides (amikacin, gentamicin, and tobramycin), nalidixic acid, and fluoroquinolones (ciprofloxacin, norfloxacin, and levofloxacin). Quinolone chromosome-encoded resistance of the FMV5825 strain was attributed to the Ser833Ile and Asp873Asn substitutions in GyrA and the Ser803Ile and Glu843Val substitutions in ParC. The qnrB2 gene was cotransferred with blaCTX-M-15, aac(6-)-Ib-cr, blaTEM-1B, and blaOXA-1 via an IncFII plasmid. The strain belonged to the B2 phylogenetic group, O25 serogroup, and ST131, identical to an intercontinental human virulent clone. This is the first report of the O25-ST131 human virulent E. coli CTX-M-15-producing clone harboring the qnrB2 and aac(6-)-Ib-cr genes in animals. It is possible that human-to-animal or animal-to-human transmission of the O25-ST131 clone has occurred.