Multiple locus variable number of tandem repeats (VNTR) analysis (MLVA) has been shown to provide a high level of information for epidemiological investigations and the follow-up of Pseudomonas aeruginosa chronic infection. In the present study, an automatized MLVA assay has been developed for the analysis of 16 VNTRs in two multiplex polymerase chain reactions (PCRs), followed by capillary electrophoresis. The result in the form of a code is directly usable for clustering analyses. This MLVA-16(Orsay) scheme was applied to the genotyping of 83 isolates from eight cystic fibrosis patients, demonstrating that the same genotype persisted during eight years of chronic infection in the majority of cases. Comparison with pulsed-field gel electrophoresis (PFGE) analysis showed that both methods were congruent, MLVA providing, in some cases, additional informativity. The evolution of strains during long-term infection was revealed by the presence of VNTR variants.