The plant pathogenic Betaproteobacterium Ralstonia solanacearum is a complex species in that most of the strains share the common characteristic of being naturally transformable. In this study, we used a new approach based on comparative genomic hybridization (CGH) on microarrays to investigate the extent of horizontal gene transfers (HGTs) between different strains of R. solanacearum. Recipient strains from phylotypes I, II and III were naturally transformed in vitro by genomic DNA from the GMI1000 reference strain (phylotype I) and the resulting DNAs were hybridized on a microarray representative of the 5120 predicted genes from the GMI1000 strain. In addition to transfer of the antibiotic resistance marker, in 8 of the 16 tested transformants, CGH on microarrays detected other transferred GMI1000 genes and revealed their number, category, function and localization along the genome. We showed that DNA blocks up to 30 kb and 33 genes could be integrated during a single event. Most of these blocks flanked the marker gene DNA but, interestingly, multiple DNA acquisitions along the genome also occurred in a single recombinant clone in one transformation experiment. The results were confirmed by PCR amplification, cloning and sequencing and Southern blot hybridization. This represents the first comprehensive identification of gene acquisitions and losses along the genome of the recipient bacterial strain during natural transformation experiments. In future studies, this strategy should help to answer many questions related to HGT mechanisms..