We examined the distribution of genes of aminoglycoside-modifying enzymes and 16S rRNA methylases in multidrug-resistant Acinetobacter baumannii to explore the association of these genes with drug resistance. Strains isolated from clinical specimens were screened using an automatic microbial identification system, and 9 aminoglycoside-modifying enzyme and 6 16S rRNA methylase genes were analyzed using polymerase chain reaction and verified by DNA sequencing. Next, sequence alignment was carried out using the Chromas software and a susceptibility test was performed using the Kirby-Bauer disk diffusion method. Genes encoding aminoglycoside-modifying enzymes were detected in all 20 strains of multidrug-resistant A. baumannii. The positive rates of aac(3')-I, aac(6')-Ib, ant(3'')-I, and aph(3')-I were 90.0, 90.0, 85.0, and 35.0%, respectively. However, genes encoding 16S rRNA methylases were not positively detected in the 20 strains of multidrug-resistant A. baumannii. The resistance of multidrug-resistant A. baumannii may be associated with aminoglycoside-modifying enzyme genes but not with 16S rRNA methylase genes.