The genetic characterization of Xanthomonas species remains a challenge. Several DNA-based techniques have been previously employed, including the analysis of the 16S rRNA and 16S-23S rRNA genes in order to differentiate and classify the Xanthomonas species. However, several species could not be distinguished in these studies, due to the high degree of conservation of these molecular markers. In order to obtain more efficient markers, and to better understand the phylogenetic relationships between the Xanthomonas species, two genes commonly found in the different species have been analyzed. The genes rpfB and atpD involved in the regulation of pathogenicity factors and in the synthesis of ATP, respectively, were amplified in Xanthomonas species and further analyzed by PCR-restriction fragment length polymorphism. Dendrograms with the data sets of the rpfB and atpD analyzed separately and combined were constructed. The results obtained revealed that several Xanthomonas species, previously grouped together, could be successfully distinguished using these markers. The results obtained herein provide an alternative method for the distinction of the Xanthomonas species and contribute to a better understanding of the genetic and phylogenetic relationships of Xanthomonas.