The xylem-inhabiting bacterium Xylella fastidiosa (Xf) is the causal agent of Pierce's disease (PD) in vineyards and citrus variegated chlorosis (CVC) in orange trees. Both of these economically-devastating diseases are caused by distinct strains of this complex group of microorganisms, which has motivated researchers to conduct extensive genomic sequencing projects with Xf strains. This sequence information, along with other molecular tools, have been used to estimate the evolutionary history of the group and provide clues to understand the capacity of Xf to infect different hosts, causing a variety of symptoms. Nonetheless, although significant amounts of information have been generated from Xf strains, a large proportion of these efforts has concentrated on the study of North American strains, limiting our understanding about the genomic composition of South American strains - which is particularly important for CVC-associated strains. This paper describes the first genome-wide comparison among South American Xf strains, involving 6 distinct citrus-associated bacteria. Comparative analyses performed through a microarray-based approach allowed identification and characterization of large mobile genetic elements that seem to be exclusive to South American strains. Moreover, a large-scale sequencing effort, based on Suppressive Subtraction Hybridization (SSH), identified 290 new ORFs, distributed in 135 Groups of Orthologous Elements, throughout the genomes of these bacteria. Results from microarray-based comparisons provide further evidence concerning activity of horizontally transferred elements, reinforcing their importance as major mediators in the evolution of Xf. Moreover, the microarray-based genomic profiles showed similarity between Xf strains 9a5c and Fb7, which is unexpected, given the geographical and chronological differences associated with the isolation of these microorganisms. The newly identified ORFs, obtained by SSH, represent an approximately 10% increase in our current knowledge of the South American Xf gene pool and include new putative virulence factors, as well as novel potential markers for strain identification. Surprisingly, this list of novel elements include sequences previously believed to be unique to North American strains, pointing to the necessity of revising the list of specific markers that may be used for identification of distinct Xf strains.