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Escherichia coli (E. coli) is a ubiquitous microorganism and a resident bacterium in the intestines of animals and humans. They are related to various intestinal and parenteral diseases. Due to the widespread and inappropriate use of antibiotics, E. coli is gaining resistance at the genetic level through horizontal gene transfer mechanisms. This study aims to decipher the antibiotic resistance pattern of clinical isolates of Escherichia coli with reference to aminoglycosides. Culturing of clinical isolates of E. coli on MacConkey and blood agar (n = 200). These isolates were identified by microscopy and biochemical examination, and confirmed at the genus level by API®20E kit. For gene confirmation, the E. coli housekeeping gene uidA was amplified by PCR. The antibacterial spectrum of the characteristic isolate was decrypted by Kirby-Bauer assay. Finally, PCR was used to screen aminoglycoside resistance determinants. According to our results, the distributions of E. coli samples were 70, 50, 10 and 70 in urine, pus, swab and stool respectively. Males (37.5%) in this study were found less infected with E. coli than females (62.5%). According to antibiotic susceptibility testing, the isolates are resistant to amikacin, ceftriaxone, cefotaxime, Ceftazidime, ciprofloxacin, Gentamicin, Moxifloxacin, Ampicillin, Cefepime, tetracycline, and Ceftaroline. 80% of the strains are resistant to Gentamicin, and 75% are resistant to amikacin. However, all isolates were sensitive to meropenem, imipenem and tigecycline. Our isolates contain 11.5% rmtB, 7% aadA1 and 8.5% aphA6 aminoglycoside resistance gene. In summary, our study describes the E. coli infection trends and resistance patterns in Islamabad, Pakistan. Further research is needed to screen other aminoglycoside resistance genes in our local environment.