A total of 75 Acinetobacter isolates resistant to all available aminoglycosides obtained from 2 Korean hospitals were studied for the genetic basis of resistance to aminoglycosides. The MIC(50) and MIC(90) of Acinetobacter baumannii isolates (n = 61) to amikacin, gentamicin, isepamycin spectinomycin, streptomycin, and tobramycin were higher than those of Acinetobacter genomic species 13TU isolates (n = 14). Genes encoding aminoglycoside-modifying enzymes, ant(3")-Ia, aac(6')-Ib, aph(3')-1a, aac(3)-Ia, and aph(3')-VI, and 16S ribosomal RNA (rRNA) methylase armA were detected. ant(3")-Ia and aac(6')-Ib were commonly detected in both Acinetobacter spp., but armA and aph(3")-Ia were only detected in A. baumannii. armA was located on the plasmids. A. baumannii isolates carrying armA were classified into 7 pulsotypes but belonged to sequence group 1. The combination of aminoglycoside-modifying enzymes is responsible for the moderate-level resistance to aminoglycosides in Acinetobacter genomic species 13TU, whereas armA is responsible for the high-level resistance to aminoglycosides in A. baumannii sequence group 1.