The most frequent Gram-negative bacterium that causes urinary tract infections is Escherichia coli, recognized as an opportunistic pathogen. E. coli strains account for more than 85% of UTI cases. This study's goal is to identify the efflux pump genes (acrAB, oqxAB, and marA) that were isolated from a variety of UTI patients under the supervision of a specialist physician at two healthcare facilities, AL-Diwaniyah General Teaching Hospital and Maternity and Pediatrics Teaching Hospital in the AL-Diwaniyah city, Iraq. The study investigated the prevalence of E. coli, which causes UTIs, which included 200 urine samples from individuals with urinary tract infections (UTIs) ranging in age from 5 to 62 years, which were tested for bacterial infection. Cultures of urine samples were isolated from 200 suspected patients with urinary tract infection (UTI) on blood and MacConkey agar and confirmed by the Vitek-2 system.  Our results show that only 50 (39%) have been positively diagnosed with E. coli out of 200 patients, and an antibiotic sensitivity test was performed using the same system. AcrAB, oqxAB, and marA are efflux pump genes expressed using real-time PCR. The study revealed that 50 (100%) of the E. coli isolates had a high rate of oqxAB gene expression (15.22%), while the ratios of acrAB and marA genes were 5.88% and 4.03%, respectively. In conclusion, the majority of E. coli isolates studied were obtained from individuals suffering from multiple drug-resistant (MDR) urinary tract infections.