Pseudomonas aeruginosa is an opportunistic pathogen that can form biofilms in the lungs and airways of cystic fibrosis (CF) patients, resulting in chronic endobronchial infection. Two clonal strains of P. aeruginosa, named type A and type B, have recently been identified and have been found to infect more than 20% of CF patients in Ontario, Canada. In this study, 4 type A and 4 type B isolates retrieved from 8 CF patients in Ontario, Canada, were characterized. All 8 isolates grew well in rich medium and formed biofilms in vitro. Antibiotic resistance profiles of bacteria grown in biofilms and planktonic culture were studied via minimal bactericidal concentration assays for tobramycin, gentamicin, and ciprofloxacin. Compared to laboratory strains of P. aeruginosa, all 8 isolates showed increased resistance to all antibiotics studied in both biofilm and planktonic assays. Gene expression analysis of mexX, representing the MexXY-OprM efflux pump, and mexA, representing MexAB-OprM, revealed that these genes were up-regulated in the 8 clinical isolates. These results suggest clonal type A and type B isolates of P. aeruginosa isolated from CF patients in Ontario, Canada, show a multidrug resistance pattern that can be partially explained as being due to the increased expression of common antibiotic efflux systems.