In summer and early autumn 2016, as a part of a country-wide survey, five Gram-negative, yellowpigmented bacterial isolates were recovered from diseased alfalfa (Medicago sativa cv. Bonab) plants in southern Iran. The isolates were negative for pectolytic activity but showed a weak amylolytic activity, and were sensitive to ≥100 mg/l, ≥0.5 g/l and ≥20 mg/l of copper sulfate, copper oxychloride, and streptomycin, respectively. All isolates were pathogenic on alfalfa as well as on common bean, faba bean, mung bean, soybean and pea. The phenotypic tests and detection with the genusspecific PCR primers established the strains as those belonging to Xanthomonas spp. Phylogenetic analysis, using the hrpB2 gene sequences, showed that all isolates belong to X. alfalfae. Multilocus sequence typing analysis, based on five housekeeping genes (fusA, gapA, gltA, lacF, and lepA) of two representative isolates, identified them as X. alfalfae subsp. alfalfae. The possible impact of this pathogen in the country is discussed, in relation to other recently reported Xanthomonads, including X. euvesicatoria and X. perforans.