Objective To investigate the prevalence and molecular epidemiology of clinical isolates of plasmid-mediated 16S rRNA methylases-producing Klebsiella pneumoniae. Methods From January 2006 and September 2007, 337 non-replicate clinical isolates of Klebsiella pneumoniae were consecutively collected from inpatients in a teaching hospital in Wenzhou, China. All of the isolates were identified by the automated microbiology systems. MICs of amikacin, gentamicin and tobramycin were determined by agar dilution method. The isolates were investigated for the presence of ESBLs by the CLSI-recommended confirmatory tests. PCR was used to detect 16S rRNA methylase genes, ESBL genes and class Ⅰ integrase gene. The homology of these isolates was analyzed by pulse-field gel electrophoresis (PFGE). Results Sixty-four ( 19. 0% ), 28 ( 8. 3% ) and 55 ( 16. 3% ) of 337 isolates were resistant to gentamicin, amikacin and tobramycin, respectively. Twenty-one (6. 2% ) isolates carried 16S rRNA methylase genes (3 for armA, 13 for rmtB, 5 for both armA and rmtB) and high-level resistant to gentamicin, amikacin and tobramycin ( MICs ≥256 mg/L). Nineteen of 21 isolates with 16S rRNA methylase genes were ESBL producers, blaCTX-M-14-like, blaCTX-M-like and blaSHV-12-like were predominant genotypes of ESBLs. The plasmids of 13 isolates were transferred into the recipients E. co/iJ53. PCR and sequence analysis revealed that blaCTX-M-14-like,blaCTX-M-15-like and blaSHV-12-like were co-transferred with the armA or the rmtB to the recipients. All transconjugants harbored intll and blaTEN-1. Of the 21 isolates, 14 patterns were obtained by PFGE. Conclusion Both horizontal gene transfer and clonal spread were responsible for the dissemination of the rmtB or the armA gene in Klebsiella pneumoniae.