Abstract: Objective To study the prevalence of 16S rRNA methylase genes in extended-spectrum-lactamascs(ESBLs)-producing Enterobacteriacea, and the correlations of 16S rRNA methylase genes with anminoglycoside resistnace. Methods Seventy-four ESBLs-producing Enterobacteriacea stains were isolated from urinary tract infections. Minimal inhibitory concentrations (MICs) of 5 aminoglycosides against the ESBLs-producing Enterobacteriacea were detected by two-fold agar dilution method. PCR amplification and DNA sequencing were used for screening and identifying 16S rRNA methylase genes. The clonality of 16S rRNA methylase gene positive ESBLs-producing Enterobacteriaceae was assessed by multilocus sequence typing (MLST). Results The bacterial resistant rates to gentamycin, netilmicin, tobramycin,amikacin and isepamicin were 93.4%, 18.4%, 13.2%, 5.3% and 5.3%, respectively. Tweenty-two out of 74 clinical isolates were 16S rRNA methylase genes positive (29.7%), including 18 armA gene and 7 rmtB gene, and 3 strains with both genes. The resistant rates of those 22 strains to gentamycin , netilmicin, tobramycin, amikacin and isepamicin were 100%, 100%, 59.1%, 18.2% and 18.2%, respectively. Among 19 E.coli isolates, seven sequence types (STs) were identified，named as ST117 (12 strains), ST2003 (2 strains), ST3843 (1 strain), ST915 (1 strain), ST844 (1 strain), ST2581 (1 strain) and ST2922 (1 strain). MLST showed that 3 K.pneumoniae isolates were nonclonal. Conclusion 16S rRNA methylase genes were widely distributed in urinary ESBLs-producing Enterobacteriacea, showing obvious relationship with the resistance to aminoglycosides. The therapy of Amikacin or Isepamicin may be considered in UTIs with 16S rRNA gene positive ESBLs-producing Enterobacteriacea.