Cystic fibrosis (CF) patients commonly suffer from continuous and recurrent lung infections caused by Pseudomonas aeruginosa, the dominant pathogen in CF airways. This study aimed to determine the integron types, gene cassettes, virulence determinants, β-lactam resistance genes, biofilm formation and alginate production in P. aeruginosa isolated from Iranian CF patients. A total of 143 P. aeruginosa isolates were obtained from CF patients. Susceptibility of isolates to different antimicrobials was evaluated by disc diffusion method. ESBL, MBL and KPC production was assessed. Congo red agar and tissue culture plates were used for evaluation of biofilm formation. Alginate production was determined using the Carbazole assay. Integrase genes, resistance determinants (ESBLs, MBLs and KPC) and genes encoding virulence factors were evaluated by PCR. All isolates were susceptible to colistin, piperacillin-tazobactam and ticarcillin; 8.4% of isolates were considered as MDR phenotype. Out of 6.3% IPM-resistant isolates, prevalence of virulence genes was as follows: lasB (100%) and plcB (100%), plcH (96.5%). Biofilm formation and alginate production ability were found in 54.5% of isolates. The prevalence of the alginate-encoding genes was 92.3%, 86.7% and 67.1% for algD, algU and algL genes, respectively. PpyR, pslA and pelA genes were detected in 98.6%, 89.5% and 57.3% of the isolates, respectively. The high prevalence of colonization in CF lungs may increase the pathogenicity of P. aeruginosa due to their adhesion and protective properties caused by biofilm- and alginate-production. LasB, plcB, plcH, exoS, toxA, algD, ppyR and pslA genes were predominant in CF P. aeruginosa strains.