<p>Modern farming methods can increase the spread of plant diseases, but whole-genome sequencing enables us to make it possible for studying on disease distribution and how the pathogen populations are varied. However, challenges remain in the efficient and cost-effective diagnosis of major pathogen bacteria species. To address this limitation, we have developed a marker system in order to assess genetic diversity in <i>Xanthomonas</i> species infecting tomato and pepper. We have utilized from the whole-genome sequence analysis and repetitive regions within the bacterial genome. Specifically, we amplified microsatellite regions using PCR, and the resulting fragments were digested with specific restriction enzymes. These fragment profiles served as molecular markers. Consequently, a more precise marker for bacterial identification was developed by combining the microsatellite and RFLP methods to differentiate Xanthomonads species. In addition, we analysed the ancestral ordering of various <i>Xanthomonas</i> species’ genomes available in NCBI using Progressive Mauve Alignment. The data revealed unique collinear regions which are characteristic for <i>Xanthomonas</i> species. These regions were also associated with digested genomic fragments used as markers, enabling the discrimination of <i>Xanthomonas</i> species which are specific for the ones infecting tomato and pepper. We propose that these findings contribute to understanding the genetic diversity of <i>Xanthomonas</i> and rapid diagnosis.</p>