Pseudomonas aeruginosa is a major opportunistic pathogen causing human infections and represents the main cause of morbidity and mortality in cystic fibrosis patients. The fluoroquinolone Ciprofloxacin is one the antibiotics most frequently used in the treatment of nosocomial infections with this bacterium. However, once established, P. aeruginosa is extremely difficult to eradicate due to the pathogen‟s ability to progress through a series of genetic and physiological changes that facilitate infection and persistence. Clinical resistance to ciprofloxacin (often accompanied with cross-resistance to other non-related antibiotics) has been reported to emerge quickly among clinical strains of P. aeruginosa. Thus, the antibiotic therapy must be carefully selected in terms of drug combination, concentration and time of exposure in order to prevent the selection and establishment of multidrug resistance strains. In this work we analyze the molecular nature of ciprofloxacin resistance for the PAO1 wild type strain and the hypermutators mutS and mutT, defective in the mismatch repair and the 8-oxoguanine repair systems respectively. To this purpuse, we sequenced the main genes known to be involved in ciprofloxacin resistance (gyrA and parC genes coding the topoisomerases target of ciprofloxacin, and nfxB coding for the represor of a multidrug efflux pump) for groups of resistant cells selected at different ciprofloxacin concentrations. Although we found some differences between the analyzed strains in terms of the resistance mechanisms and the mutational spectra, all of them showed a high percentage of gyrA mutations at high ciprofloxacin concentration, while the nfxB gene was mainly mutated in the groups of cells selected at low drug concentration. These nfxB mutants showed cross-resistance to other antibiotics such as erythromycin, zwitterionic cephems and trimethoprim, in contrast to those solely mutated in the topoisomerase genes. In this context, we discuss the influence of the exposure to low and high doses of ciprofloxacin on the emergence of multidrug resistant strains, both in a wild type and hypermutator backgroun