Antibiotic resistance is more challenging in third world countries due to irrational use of antibiotics and poor antimicrobial resistance surveillance. The current study is aimed to determine the molecular characterization and mutational analysis of antibiotic resistant genes of E. coli isolated from UTIs patients. A total of 112 E. coli isolates were recovered from UTI suspected patients from Khyber Teaching Hospital (KTH), Peshawar. The collected samples were identified phenotypically by API-10S strips and confirmed by Polymerase Chain Reaction. The selected antibiotic resistant genes were detected by PCR and subsequently sequenced by Next Generation Sequencing. The results of antibiogram revealed that the E. coli isolates were resistant to various antibiotics; Ampicillin, Cotrimoxazole and Ciprofloxacin while showed good results against Tigecycline, Meropenem and Cefoperazone-Sulbactam. Molecular analysis showed that 72 isolates were HIGHLIGHTS• Mutational analysis of antibiotic resistant genes of E. coli in UTI Patients.• These genes were detected by PCR and sequenced using NGS.• E. coli isolates were positive for GyrA gene, GyrB gene, tetB and tetR gene.• The novel mutation was detected in gyrB gene at codon554.