Introduction: 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.