Background: Escherichia coli is the most important as well as the most common bacteria causing urinary tract infections (UTIs) and its resistance to common antibiotics is increasing. Extended-spectrum beta-lactamase (ESBL) producerE. coli strains can resist against the third-generation and fourth-generation cephalosporins.Objectives: This study aimed to evaluate the resistance profile of E. coli isolated from patients with UTIs referred to Imam Khomeini and Baqiyatallah Hospitals, Tehran, Iran, through phenotypic and molecular methods.Materials and Methods: During 2010-2011, 180 urine samples of patients with UTIs from Imam Khomeini and Baqiyatallah Hospitals were collected. Based on the standard bacteriologic tests, E. coli isolates were identified. Resistance to common antibiotics was tested by the Kirby-Bauer method and reconfirmed by determining minimum inhibitory concentration (MIC) through microdilution method. Further phenotypic double-disk synergy test (DDST) was performed to screen the ESBL producer strains. Resistance genes related to ESBL andqnrA were evaluated by Polymerase chain Reaction (PCR).Results: A total of 100 E. coli strains were examined by antibiogram and the rates of resistance to the tested antibiotics were as follows: 100% to penicillin and amoxicillin, 77% to amoxicillin-clavulanic acid, 72% to ceftazidime, 69% to cefotaxime, 47% to cefoxitin, 46% to ceftriaxone, 43% to cephalexin, 27% to aztreonam, 53% to nalidixic acid, 51% to ciprofloxacin, and 2% to imipenem. The MIC to ciprofloxacin, cefazolin, and ceftriaxone were ≥ 0.249, ≥ 0.508, and ≥ 0.044, respectively. Moreover, 20% of E. coli isolates were ESBL-producing isolates by DDST. The frequency ofbla CTX-M, bla TEM, bla SHV, and qnrA genes was 87%, 82%, 65%, and 39%, respectively. Conclusions: Considering the high prevalence of ESBL genes (bla CTX-M, 87%; and bla TEM, 82%), fluoroquinolones may be used as an alternative drug in treatment, although resistance to this family is increasing as well. As a result, this increasing trend should be prevented using appropriate guidelines for prescription.