Treatment with antibiotics leads to the selection of isolates with increased resistance. We investigated if evolution towards resistance was associated with virulence changes, in the context of P. aeruginosa ventilator-associated pneumonia (VAP). Four patients were selected because they had multiple VAP episodes during short periods (12 days to 5 weeks), with emergence of resistance. We performed whole-genome sequencing of 12 P. aeruginosa from bronchoalveolar lavages or blood culture (3 isolates per patient). Production of quorum sensing-dependent virulence factors, serum resistance, cytotoxicity against A549 cells, biofilm production, and twitching motility were studied. Each patient was infected with a unique strain. For all patients, resistance development was explained by genetic events in ampD, mexR or oprD. Additional variations were detected in virulence- and/or fitness-associated genes (algB, gacA, groEL, lasR, mpl, pilE, pilM, rhlR) depending on the strain. We noticed a convergence towards quorum sensing deficiency, correlated with a decrease of pyocyanin and protease production, survival in serum, twitching motility and cytotoxicity. In one patient, changes in pilM and pilE were related to enhanced twitching. We show that the emergence of resistance in P. aeruginosa is associated with virulence modification, even in acute infections. The consequences of this short-term pathoadaptation need to be explored.