Clinical isolates of Acinetobacter baumannii have a tendency to develop antimicrobial resistance against commonly prescribed antimicrobial agents, including aminoglycoside agents, particularly in hospitalized patients worldwide. Resistance mechanisms of the bacterium to aminoglycosides are diverse and commonly involve production of aminoglycoside-modifying enzymes and efflux systems. The aim of this study was to investigate the frequency of gene encoding aminoglycoside-modifying enzymes and expression level of adeB efflux gene in A. baumannii isolates recovered from burn wound colonization. A total of 47 clinical isolates of A. baumannii were obtained from burned patients admitted to the Burns Teaching Hospital, Tehran, in 2018. Standard antimicrobial susceptibility screening was performed to determine resistance pattern. A polymerase chain reaction (PCR) assay was performed to determine aminoglycoside-modifying genes ACC(6'), aph(3')-Via, aph(3')-IIb, aadA1, aphA1 and aph6. Semi-quantitative RT-PCR was also carried out to quantify the expression level of the adeB gene. According to the results of the present study, the acc(6') was the predominant aminoglycoside-modifying enzyme gene (80.9%), followed by aph(3')-via, aph6, aph(3')-IIb and aphA1, which was detected in 59.6%, 42.6%, 14.9% and 14.9% of isolates, respectively. None of the A. baumannii isolates harboured the aadA1 gene. The up regulation of adeB gene expression was observed in 63.8% of strains. Moreover, we indicated that there is a relationship between adeB expression and high resistance to gentamicin. Our results revealed that aminoglycoside resistance could be explained by the production of one or a combination of known aminoglycoside-modifying enzymes rather than overexpression of adeB.