Horizontal gene transfer (HGT) is a major driving force of evolution and is also likely to play an important role in the threatening emergence of novel pathogens, especially if it involves distantly related strains with substantially different pathogenicity. In this study, the impact of natural transformation on pathogenicity in six strains belonging to the four phylotypes of the plant-pathogenic bacterium Ralstonia solanacearum was investigated. The study focused on genomic regions that vary between donor and recipient strains and that carry genes involved in pathogenicity such as type III effectors. First, strains from R. solanacearum species complex were naturally transformed with heterologous genomic DNA. Transferred DNA regions were then determined by comparative genomic hybridization and polymerase chain reaction sequencing. We identified three transformant strains that acquired large DNA regions of up to 80 kb. In one case, strain Psi07 (phylotype IV tomato isolate) acquired 39.4 kb from GMI1000 (phylotype I tomato isolate). Investigations revealed that i) 24.4 kb of the acquired region contained 20 new genes, ii) an allelic exchange of 12 genes occurred, and iii) 27 genes (33.4 kb) formerly present in Psi07 were lost. Virulence tests with the three transformants revealed a significantincrease in the aggressiveness of BCG20 over its Psi07 parent on tomato. These findings demonstrate the potential importance of HGT in the pathogenic evolution of R. solanacearum strains and open new avenues for studying pathogen emergence.