ntroduction Resistance to fluoroquinolones (FQ) in uropathogenic Escherichia coli (UPEC) has emerged as a growing problem. Chromosomal mutations and plasmid-mediated quinolone resistance (PMQR) determinants have been implicated. Data concerning the prevalence of these determinants in UPEC in our hospital are quite limited. Purpose To investigate the occurrence and genetic determinants of FQ resistance in UPEC isolated from urinary tract infection (UTI) cases in Zagazig University Hospitals. Patients and Methods Following their isolation, the identification and susceptibility of UPEC isolates were performed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometer (MALDI-TOF MS). FQ resistance was detected by the disc diffusion method. Ciprofloxacin minimal inhibitory concentration (MIC) was determined using E-test. Chromosomal mutations in the gyrA gene were detected using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and for detection of PMQR, a couple of multiplex PCR reactions were used. Results Among a total of 192 UPEC isolates, 46.9% (n=90) were FQ resistant. More than half of the isolates (57.8%) exhibited high-level ciprofloxacin resistance (MIC > 32 µg/mL). Mutations in gyrA were detected in 76.7% of isolates, with 34.4% having mutations at more than one site. PMQR determinants were detected in 80.1% of UPEC isolates, with aac(6ʹ)-Ib-cr gene being the most frequent found in 61.1% of isolates. Conclusion There is a high prevalence of both gyrA mutations and PMQR determinants among UPEC isolates in our hospital which contribute to high-level ciprofloxacin resistance, a finding that may require the revision of the antibiotics used for empirical treatment of UTI. Keywords: gyrA mutations, qnr determinants, uropathogenic E. coli, ciprofloxacin resistance