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Background: TEM, PER, and VEB are extended-spectrum betalactamase (ESBL) enzymes that are capable of hydrolyzing penicillins, cephalosporins, and aztreonam. The aim of this study was to determine the prevalence of ESBL producing E.coli and molecular evaluation of TEM, PER, and VEB β-lactamases among E.coli strains. Materials and Methods: A total of 200 clinical strains of E.coli were isolated from clinical specimens and their antibiotic susceptibility was determined by disk diffusion method. ESBL production was determined using the combined disk method with CAZ and CTX with clavulanic acid and alone. Minimum concentration inhibition (MIC) for CAZ and CTX with clavulanic acid and alone was determined by agar dilution method. Finally, PCR with specific primers was used for determining the presence of blaTEM, blaPER, and blaVEB genes. Results: Combined disk method confirmed 94 strains (47%) to be ESBL producing E.coli. Of the 94 ESBL producing strains, 36 samples had MIC=16, 44 samples had MIC between 32-256, and 10 samples had MIC≥512 for ceftazidime, whereas 8 samples had MIC=16, 68 samples had MIC between 32-256, and 21 samples had MIC≥512 for cefotaxime. The frequency of TEM was 44% however, blaPER and blaVEB genes were not detected by PCR among ESBL producing isolates. Conclusion:The results indicated that the high percentage of ESBL producing E.coli is 47% and PCR method showed a high frequency of TEM enzyme, but PER and VEB betalactamase were not found among them.