Abstract Background: Pseudomonas aeruginosa is one of the most prevalent Gram-negative microbes associated with serious and fatal nosocomial infections. It is routine to use aminoglycosides as a treatment for P. aeruginosa infections. Objectives: The dissemination of aminoglycoside resistance offers a major challenge to the treatment of life-threatening infections caused by these bacteria. Aim of Study: Determination of aminoglycoside-modifying enzymes and 16S ribosomal RNA methylases in extensive-drug resistant (XDR) P. aeruginosa isolates. Materials and Methods: From November 2021 to August 2022, a total of 200 samples were collected in this cross-sectional study including burns (n = 80, 40%), wounds (n = 66, 33%), and diabetic foot ulcers (n = 54, 27%) from admitted patients to Al-Diwaniyah Burn Center and Al-Diwaniyah Teaching Hospital. The identification and antibiotic susceptibility profile of P. aeruginosa were done by vitek2 compact system. The isolates were subjected to polymerase chain reaction assays with specific primers for ant(4’)-IIa, ant(4’)-IIb, acc(6’)-Ia, aph(3’)-IIb, rmtA, and rmtD. Results: The recovery rate of P. aeruginosa isolates was (n = 50, 25.0%) from the clinical samples. Antibiotic-susceptibility patterns demonstrated that 18% of the isolates were multi-drug resistant and 22 (44%) were XDR. The XDR isolates were resistant to all 14 antibiotics related to the seven antibiotic classes tested in this study. The prevalence of aminoglycoside resistance genes among XDR isolates is ant(4’)-IIa (22.7%), (4’)-IIb (27.3%), acc(6’)-Ia (18.18%), aph(3’)-IIb (100%), rmtA (36.36%), rmtD (36.36%), mexZ and parR (100%). Conclusion: Increased resistance to aminoglycosides in Al-Diwaniyah Hospitals serves to highlight how critical this issue is when treating multidrug-resistant P. aeruginosa infections that are life-threatening. All the resistance isolates harbored aph(3’)-IIb gene.