Objective To investigate the prevalence of 16S rRNA methylase genes, armA and rmtB, which mediate high level aminoglycoside resistance, in gram-negative bacteria isolated from 2 hospitals in Dalian and study the mechanism of aminoglycoside resistance.Methods A total of 134 amikacin-resistant clinical isolates of gram-negative bacteria were collected. Two 16S rRNA methylase genes, armA and rmtB, were identified by PCR-based assays. PCR products were extracted for DNA sequencing analysis. armA and rmtB gene mapping were conducted by plasmid extraction, conjugation and transformation. The MICs of amikacin, gentamicin and tobramycin were determined for the positive isolates, transconjugants and the resultant strain of transformation using agar dilution technique. Results Overall armA was identified in 21 strains of Acinetobacter baumannii, rmtB in 5 strains of Escherichia coli and 5 strains of Klebsiella pneumoniae. Plasmid extraction and conjugation experiments were only successful for rmtB-positive isolates. Transconjugant and DH5a (pMDarmA) exhibited high-level resistance to aminoglycosides.Conclusions The 16S rRNA methylase genes, armA and rmtB are identified in Dalian. armA gene is identified in A. baumannii. rmtB gene is located on the plasmid of E. coli and K. pneumoniae. armA and rmtB can induce high-level resistance to aminoglycosides.