Objective To investigate the genotypes of aminoglycoside-modifying enzymes (AMEs) produced by Pseudomonas aeruginosa isolated from Huaibei Miner's General Hospital.Methods Minimum inhibitory concentration of 36 strains of P. aeruginosa to 3 aminoglycoside antibiotics was determined. AME genes were amplified by polymerase chain reaction (PCR). Results The P. aeruginosa isolates were resistant to multiple antibiotics. Most (62.2% to 81.1%) were resistant to aminoglycosides. And 27 strain (75.0%) carried one or more types of AME genes, including ant(3″)-Ⅰ (63.9%), aac(6')-Ⅱ (58.3%), aac(6')-Ⅰ (50.0%), aac(3)-Ⅱ (38.9%) and ant(2″)-Ⅰ (36.1%). The aac(3)-Ⅰ, aac(3)-Ⅲ, aac(3)-Ⅳ, aph(3')-Ⅳ genes were not identified.Conclusions The study indicates that the P. aeruginosa isolates in Huaibei are multi-resistant to aminoglycoside antibiotics. The prevalence of AMEs-positive strains is high.