The aim of this study was to assess the incidence and molecular epidemiology of multidrug-resistant (MDR) Pseudomonas aeruginosa in our university hospital. Analysis included antimicrobial susceptibility profiling, bla gene identification and pulsed-field gel electrophoresis (PFGE). During the one-year study, 654 patients had at least one sample that tested positive for P. aeruginosa, of whom 38 (5.8%) were colonised or infected with an MDR isolate, giving an incidence of 0.1 patient per 1000 patient-days. The 38 non-duplicate isolates yielded 12 different PFGE patterns, three of which included isolates from four patients and one of which included isolates from 15 patients. Two isolates produced acquired extended-spectrum β-lactamase (one OXA-14 and one OXA-28). Genotyping showed that cross-transmission was responsible for about 70% of MDR P. aeruginosa cases although spatio-temporal analysis failed to demonstrate when this might have occurred for most cases. The major epidemic and the three main micro-epidemic clones were already present in our hospital with a more susceptible phenotype. It is likely that some P. aeruginosa clones are endemic in our hospital and that, within these clones, MDR isolates emerge under antibiotic pressure. Our results indicate that cross-transmission plays a major role in the spread of MDR P. aeruginosa and suggest that priority should be given to the improvement of standard hygienic precautions.