Introduction: Pseudomonas aeruginosa is a worldwide nosocomial infection that disrupts hospitalized patient's recovery. Use of the proper antipseudomonas antibiotic therapy is an expected challenge among health care providers. Methods: This is a cross-sectional in-vitro study which was conducted in Isfahan in 2016-2017. After selection of the patients with P. aeroginusa infection and isolation of bacteria, the presence of AME coding genes such as aac(6')-II and ant(2'')-I was surveyed by PCR method. Result: From 350 patients with P.aeruginosa infection, 100 samples were obtained. About half of the strains were taken from urine samples and respiratory tract swap. 93% of Pseudomonas aeruginosa strains were resistant to at least one antibiotic. The highest antibiotic resistance was dedicated to Tobramycin and Meropenem. However, all strains were sensitive to Colistin. In regard to AME genome, 13 specimens had ant(2'')-I and 4 strains had aac(6')-II genes. aac(6')-II gene caused 75% resistance to Amikacin, Tobramycin, and Gentamycin while ant(2'')-I gene posed Tobramycin and Gentamycin resistance. Conclusion: Overall, there was a high resistance ratio to various aminoglycosides, B-lactams, and fluoroquinolones isolated from infected patients. Less than 20% of strains demonstrated AME genes of aac(6')-II and ant(2'')-I.