Objectives: Escherichia coli are resistant to β-lactam antibiotics due to the acquisition of plasmids that encode broad-spectrum β-lactamases. Extended-spectrum β-lactamases (ESBLs) have particular importance in antimicrobial therapy. The current study aimed at determining the frequency and role of SHV, CTX-M, and IMP genes in resistance to ceftriaxone, imipenem, and piperacillin antibiotics in E. coli isolated from patients with urinary tract infection (UTI). Methods: The study population included outpatients and inpatients of two genders in all ages with UTI. A total of 270 non-duplicate E. coli stains producing ESBL from Velayat Hospital, Damghan city, Semnan province, Iran were isolated using screening and doubledisc phenotypic test. All isolates were screened to detect SHV, CTX-M, and IMP genes using polymerase chain reaction (PCR). Results: In total, 120 (44.4%) samples were positive for ESBLs including 30% CTX-M, 27.5% SHV, and 0% IMP genes. The obtained results showed that the responses of male and female patients to the examined antibiotic were different. The distribution of underlying diseases (diabetes, kidney stones, obstructive disease, congenital anomalies, and hospitalization history) was different in patients with UTI. It was observed that after hospitalization history, diabetes had the highest frequency in female (31.08%) and male (27.59%) patients with UTI. Conclusions: However, bacterial strains producing ESBLs such as E. coli should be considered as a major threat to public health due to the risk of resistant to broad-spectrum cephalosporins. Therefore, to prevent resistance to antibiotics, indiscriminate administration of such agents should be avoided.