Background: Humans can develop both acute and chronic infections from Pseudomonas aeruginosa, a bacterium that is a prominent cause of nosocomial infections, as well as respiratory, urinary tract, burn, wound, ear, eye, and bloodstream infections. Objectives: This study aimed to identify antibiotic resistance in P. aeruginosa strains obtained from hospitalized patients and assess the frequency of virulence genes. Methods: In this cross-sectional study, 86 strains of P. aeruginosa were isolated from patients in various hospital wards in Shiraz and Yasuj in 2021. Antibiotic susceptibility was tested using the disk diffusion method, while the frequency of virulence genes (aprA, phzM, phzS) and antibiotic resistance genes (blaGES, blaSPM, blaVIM) was evaluated using PCR and specific primers. Results: The P. aeruginosa isolates showed the highest antibiotic resistance to imipenem and the lowest resistance to piperacillin-tazobactam. PCR amplification of aprA, phzM, and phzS genes indicated that aprA had the highest frequency among the isolates, with a prevalence of 98.8%. The frequencies of phzM and phzS were 96.5% and 91.9%, respectively. Conclusions: This study revealed a high prevalence of the virulence genes aprA, phzM, and phzS in P. aeruginosa isolates. Given the bacterium’s resistance to many antibiotics, clinicians should exercise caution when prescribing antibiotics, particularly for infections caused by P.aeruginosa.