Background and Objective: Staphylococcus aureus is a major human pathogen responsible for a wide range of clinical infections. While aminoglycoside antibiotics are vital for treating staphylococcal infections, resistance to these agents is an increasing global concern. This study aimed to determine the prevalence of aminoglycoside-modifying enzyme genes [aph-3 and armA] in clinical isolates of S. aureus from various hospitals in Khartoum State. Materials and Methods: A cross-sectional study was conducted between May and August 2022. Seventy-two [72] S. aureus isolates were obtained from clinical specimens, including wound swabs, blood, and aspirated fluids. The isolates were identified using standard biochemical tests. Antimicrobial susceptibility testing for amikacin and gentamicin was performed using the disk diffusion method. All 72 isolates were analyzed via multiplex polymerase chain reaction [PCR] to detect the presence of the aph-3 and armA resistance genes. Results: Of the 72 S. aureus isolates, 35 [48.6%] were positive for at least one resistance gene. Specifically, the aph-3 gene was detected in 34 [47.2%] isolates [including one isolate carrying both genes], while the armA gene was detected in 2 [2.8%] isolates. Phenotypic resistance was found to be higher for amikacin than for gentamicin. Statistical analysis showed no significant association between the presence of these genes and patient gender, age, or specimen type [p > 0.05]. Conclusion: This study concludes that the aph-3 gene is the predominant aminoglycoside resistance marker among S. aureus isolates in Khartoum State. The low prevalence of the armA gene suggests it is currently a less common mechanism in this region. The high frequency of resistance to amikacin underscores the need for continuous molecular surveillance to guide effective antimicrobial therapy.