Resistance of clinical strains of Pseudomonas aeruginosa to aminoglycosides can result from production of transferable aminoglycoside-modifying enzymes, of 16S rRNA methylases, and/or mutational derepression of intrinsic multidrug efflux pump MexXY(OprM). The present study reports on characterization of a new type of mutants 4- to 8-fold more resistant to 2-deoxystreptamine derivatives (e.g., gentamicin, amikacin, tobramycin), as compared with wild-type strain PAO1. The genetic alterations of three in vitro mutants were mapped on fusA1, and found to result in single amino acid substitutions in domains II, III and V of elongation factor G (EF-G1A), a key component of translational machinery. Transfer of the mutated fusA1 alleles into PAO1 reproduced the resistance phenotype. Interestingly, fusA1 mutants with other amino acid changes in domains G, IV and V of EF-G1A were identified among clinical strains with decreased susceptibility to aminoglycosides. Allelic exchange experiments confirmed the relevance of these latter mutations and of three other previously reported alterations located in domains G and IV. Pump MexXY(OprM) was found to partly contribute to the resistance conferred by the mutated EF-G1A variants, and to have additive effects on aminoglycosides MICs when mutationally up-regulated. Altogether, our data demonstrate that cystic fibrosis (CF) and non-CF strains of P. aeruginosa can acquire a therapeutically significant resistance to important aminoglycosides, via a new mechanism involving mutations in elongation factor EF-G1A.