Background: The problem of antibiotic resistance has become worldwide health issue among hospitalized patients in developing countries, where the inappropriate usage of antibiotics is common. The widespread occurrence of multidrug resistant strains (MDRS) of Escherichia coli (E. coli) and klebsiella pneumoniae (K. pneumoniae) is currently considered avital medical challenge. The identification of ESβL genes and their MDRS in widely isolated uropathogens may provide a helpful tool for choosing effective antibiotic therapy and clinical improvement. Objective: The aim of this study is to determine the prevalence of (bla.TEM, bla.CTX-M, and bla.SHV) β-lactamase DNAs in E. coli and K. pneumoniae, and MDRS of uropathogens E. coli and k. pneumoniae isolated from patients urine UTI diagnosed who were admitted to the Urology & Nephrology Center. Methodology: Clinical E. coli and K. pneumoniae isolates were isolated from urine specimens of UTI diagnosed patients. Microbiological techniques were utilised to identify the isolates. Antibiotics susceptibility analysis was done utilising the Vitek2 system (bioMérieux, Marcy l'Etoile, France). The double disc diffusion technique (DDDT) was used for detecting phenotypic ESBL in isolates. Using the traditional PCR technique, the ESBL genes were identified. Results: Antibiotic resistance of E. coli's isolates to commonly used antibiotics was high (88.2% resistant to Amoxicillin/clavulanate, 83.5 % were resistant to Trimethoprim-sulfamethoxazole) while minimum resistance appeared against Amikacin (AK) 10.5% and Imipenem (IP) 14%. For isolates of K. pneumoniae, the resistance was higher for nitrofurantoin, ceftazidime, cefotaxime, ciprofloxacin 96.6%, 83.3 %,83.3%, 80% respectively .In our study, out of 115 isolates, E. coli and K. pneumoniae isolates, 81 (70.43%) were phenotypically positive for ESβL by Combination Disc Test (CDT). The most commonly molecularly detected ESBL genes were TEM 80% of K. pneumoniae isolates and 77.6% of E. coli isolates .followed by SHV 70% of K. pneumoniae isolates and 34.1% of E. coli isolates and finally, CTXM 67% of isolates of E. coli and 63.3% of isolates of K. pneumoniae. Conclusions: The current study concluded that a high rate of resistance has been developed in uropathogens with empiric antibiotic treatment. It emphasized the alarming role of β-lactamases, especially ESßLs in antibiotic