Efflux pumps play an important role in the emergence of antibiotic-resistant Pseudomonas aeruginosa strains. The present study aimed to assess the expression of the MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY-OprM efflux pumps in carbapenem-resistant and multidrug-resistant (MDR) P. aeruginosa strains isolated from clinical specimens between June 2019 and January 2022 in Ardabil city. The presence of efflux pump-encoding genes, i.e. mexA, mexC, mexE, and mexY, was assessed using the polymerase chain reaction (PCR) technique in 48 carbapenem-resistant and MDR P. aeruginosa strains. Real-time reverse transcription PCR was employed to evaluate the expression levels of mexA, mexC, mexE, and mexY genes. All 48 carbapenem-resistant and MDR P. aeruginosa strains harbored efflux pump-encoding genes including mexA, mexC, mexE, and mexY according to the PCR results. Overexpression of the MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY-OprM efflux pumps was detected in 75% (n = 36), 83.3% (n = 40), 10.4% (n = 5) and 41.6% (n = 20) of the clinical isolates of P. aeruginosa, respectively. This study revealed that the presence and overexpression of efflux pumps are associated with the emergence of carbapenem-resistant and MDR P. aeruginosa strains. Therefore, research on efflux pump inhibitors of P. aeruginosa will be a worthwhile endeavor to increase the clinical efficiency of available antibiotics and prevent ensuing treatment failure.