Multidrug resistance is an escalating worldwide public health problem that threatens millions of people every year. Multidrug resistant uropathogenic Escherichia coli is a major cause of urinary tract infections which has contributed to the rising concern of selecting effective antibiotics against UTIs. This study was aimed at detection of AcrA-AcrB-TolC Efflux Pump Genes in locally isolated E.coli and investigating the presence of mutation in efflux pump repressor AcrR gene. Urine samples were collected from patients with urinary tract infection attending Baghdad Medical Hospital during the period from November 2021 to January 2022. Out of those samples, 100 showed the growth of uropathogenic E.coli. Molecular detection of AcrAB-TolC genes by polymerase chain reaction showed that both MDR and antibiotic-susceptible E. coli isolates possessed the chromosomal AcrAB-TolC. Mutation in the repressor AcrR was investigated by ARMS PCR and the results showed that mutation was found only in multidrug resistant uropathogenic E.coli suggesting that the mutation in the repressor AcrR gene may contributes to antibiotic resistance in UPEC.