Bacterial leaf spot of tomato and pepper (BLS), an economically important bacterial disease caused by four species of Xanthomonas [(X. euvesicatoria (Xe), X. vesicatoria (Xv), X. gardneri (Xg) and X. perforans (Xp)], is a global problem and can cause over 50% crop loss under unfavorable conditions; among the four species, Xe and Xv are prevalent worldwide. Characterization of the pathogens is crucial for management and regulatory purposes. In this study, we performed MLSA with 6 genes (dnaA, hrcN, gyrB, gapA, hmbs and pdg) to determine phylogenetic relationships within and among the four species using 77 strains from different serological groups and diverse geographical locations and also determined their phylogenetic positions relative to other xanthomonads. Both antibody and MLSA data showed that Xv was clearly separated from Xe and that the latter strains were remarkably clonal even though they originated from distant geographical locations. The Xe strains formed two separate phylogenetic groups; Xe group A consisted only of tomato strains whereas Xe Group B included strains from tomato and pepper. In contrast, the Xv group showed greater heterogeneity. Some Xv strains from South America were closely related to strains from California while others grouped closer to a strain from Indiana and more distantly to a strain from Hawaii. Using this information molecular tests can now be devised to track distribution of clonal populations that may be introduced into new geographic areas through seeds and other infected plant materials.