Urinary tract infection (UTI) is a multifactorial disease with a range of pathogenic microbes as causative agents including parasites, fungi and bacteria. However, the main causative pathogen in UTIs is bacteria. The UTI is one of the common reasons for nosocomial infection in the community. The current study aimed to determine the prevalence of UTIs followed by investigating the most effective antibiotics and the amplification of β-lactamase (bla-TEM) gene in isolated bacteria. The Present study was conducted in the tertiary care hospitals of various areas in Peshawar, Pakistan Mid-stream urine samples were collected from UTI patients. Inoculation of entire samples on MacConkey and Blood agar followed by urease, catalase test for gram-positive and citrate, and indole test for gram-negative bacteria was done. Culture sensitivity analysis for various antibiotics was done according to CLSI guidelines. DNA extraction and molecular characterization of bla-TEM resistance genes was also performed using specific primers by Polymerase Chain Reaction (PCR) technique. 117/531 (22.03%) samples were positive for UTI. Among these, most of the samples belong to female population 64.9%. In the age-wise distribution, the middle to older age group has the highest prevalence of UTI. 117 isolates separated with E. coli (62.4%) followed by Klebsiella spp (11.1%) and 7.7% of Pseudomonas as the most prevalent urinary tract infectious microbes. Antibiotic susceptibility showed gentamicin and meropenem as the most resistive antibiotics. Meropenem and ciprofloxacin were the most sensitive antibiotics. Molecular characterization showed 69/117 (58.9%) and 61/117 (52.1%) sample amplification for β-lactamase and aminoglycoside-resistant genes, respectively. It is concluded that UTI is a serious multi-pathogenic problem prevailing in District Peshawar. Most of the uropathogens show resistance towards different antibiotics based on the resistance genes detected in them.