Background: Antibiotic resistance is among the major threats to health systems worldwide. Antibiotic resistance is often associated with efflux pumps, that distinguish many antibiotics are called Multidrug Resistance (MDR) efflux pumps. Aim: investigate the prevalence of Escherichia coli bacteria that cause urinary tract infection by identifying the gene (papE) and identifying the efflux pump genes (acrB, TolC , mdfA ) using the polymerase chain reaction. Methods: 200 samples were obtained from the urine of patients with urinary tract infections from Baquba Teaching Hospital and from Al-Batoul Maternity Hospital in Diyala Governorate / Iraq during the period from January to April 2022. The nucleic acid DNA of the bacterial isolates under study was extracted and then the polymerase chain reaction (PCR) was performed through the use of specialized primers the gene (acrB, mdfA, TolC) . Results: The results of antibiotic sensitivity showed that all isolates were 100% resistant to each of Ampicillin and Augmentin, 98% Amikacin, 96% Tetracycline, 82% to Cefepime, and 50% to the results of the study showed that all bacterial isolates (20) isolates contain the gene (mdfA) 100%, and 19 isolates out of a total of 20 isolate targeting 95% contain both gene TolC and acrB. Conclusion: Most of the isolates of Escherichia coli bacteria under study, isolated from patients with urinary tract infections (UTIs) with multiple antibiotic resistance (MDR), and all bacterial isolates possessed the gene (mdfA) in a percentage of (100%), while the genes (TolC, acrB ) were found in a percentage(95%).