A multilocus sequence analysis (MLSA) was performed on five housekeeping genes (fusA, gapA, gltA, lacF and lepA) of 22 Xanthomonas euvesicatoria strains recently isolated from alfalfa, pepper and tomato plants in Iran. In addition, 161 strains isolated worldwide from pepper, poinsettia, rose and tomato plants were included in the analysis. All X.euvesicatoria pv. perforans isolates from tomato plants in Iran clustered in a monophyletic group, although five MLSA haplotypes were detected among them. The Iranian tomato strains presented 10 nucleotide differences in the lepA gene sequences compared to the known worldwide population of X.euvesicatoria pv. perforans. Statistical analyses revealed a recombination event that had occurred in the lepA gene of the strains isolated from tomato in Iran. BOX-PCR analysis confirmed the inclusion of Iranian tomato strains within X.euvesicatoria pv. perforans. Furthermore, X.euvesicatoria pv. euvesicatoria strains isolated from pepper in Iran differed in one nucleotide in the lepA gene sequence from the known worldwide population of the pathovar, and clustered in a group containing strains isolated in Nigeria. The strains isolated from alfalfa in Iran clustered with the type strain of X.euvesicatoria pv. alfalfae. Altogether, the results reveal the existence of a phylogenetically novel population of X.euvesicatoria pv. perforans in Iran which needs further in-depth analysis to pinpoint the epidemiological impact of these strains.