Tobramycin and colistin represent 2 standard antimicrobial agents in the treatment of cystic fibrosis (CF) patients who are chronically colonized with Pseudomonas aeruginosa. In this study, we determined the rate of resistance to tobramycin and colistin in 1844 isolates of P. aeruginosa obtained from 22 CF patients under alternate therapy with inhaled tobramycin and colistin. Resistance to tobramycin was observed in 27.5% of isolates. In contrast, all isolates were susceptible to colistin. Molecular typing of selected isolates suggested that only 1 clone occurred over time in each patient. To conclude, resistance to tobramycin in P. aeruginosa isolates from CF patients under antimicrobial therapy may occur while colistin resistance remains uncommon.