The rpoB gene was evaluated as an alternative molecular marker for the differentiation of Xanthomonas species and in order to understand better the phylogenetic relationships within the genus. PCR-RFLP experiments using HaeIII allowed differentiation of Xanthomonas species, particularly those that affect the same plant host such as Xanthomonas albilineans and X. sacchari , pathogenic to sugar cane, Xanthomonas cucurbitae and X. melonis , which cause disease in melon, and Xanthomonas gardneri , X. vesicatoria and X. euvesicatoria / X. perforans , pathogenic to tomato. Phylogenetic relationships within the genus Xanthomonas were also examined by comparing partial rpoB gene sequences (612 nt) and the Xanthomonas species were separated into two main groups. Group I, well supported by bootstrap values of 99 %, comprised X. euvesicatoria , X. perforans , X. alfalfae , X. citri , X. dyei , X. axonopodis , X. oryzae , X. hortorum , X. bromi , X. vasicola , X. cynarae , X. gardneri , X. campestris , X. fragariae , X. arboricola , X. cassavae , X. cucurbitae , X. pisi , X. vesicatoria , X. codiaei and X. melonis . Group II, again well supported by bootstrap values of 99 %, comprised X. albilineans , X. sacchari , X. theicola , X. translucens and X. hyacinthi . The rpoB gene sequence similarity observed among the species in this study ranged from 87.8 to 99.7 %. The results of PCR-RFLP of the rpoB gene indicated that this technique can be used for diagnosis and identification of most Xanthomonas strains, including closely related species within the genus. However, species that showed identical profiles could be differentiated clearly only by sequence analysis. The results obtained in our phylogenetic analysis suggested that the rpoB gene can be used as an alternative molecular marker for genetic relatedness in the genus Xanthomonas . The results of PCR-RFLP of the rpoB gene indicate that this technique can be used for diagnosis and identification of closely related species within the genus, representing a rapid and inexpensive tool that can be easily standardized between laboratories.