Forage plants are particularly important in economic terms and are the main ingredients in animal feed. Alfalfa is a forage plant of the Leguminosae family, and is one of the most important forage crops in Iran and the world. However, several pest and pathogen including alfalfa weevil (Hypera postica), and diseases caused by fungal and oomycete pathogens, such as Phytophthora root and stem rot, and alfalfa powdery mildew have been reported to affect alfalfa production in Iran. In summer and early autumn 2016, alfalfa (Medicago sativa cv. Bonab) plants with suspected bacterial disease symptoms were observed in Fars and Kerman provinces. Symptomatic plants showing leaf spot symptoms were collected and brought to laboratory for further studies. The samples were surface sterilized with dipping in a 0.5 % sodium hypochlorite solution, washed twice by sterile distilled water and dried on the paper tissue. Isolations were performed by cutting the marginal tissues of the visible lesions on leaves and the tissues were ground in sterile distilled water. A loopful of the resulting suspensions was streaked on Nutrient agar (NA) medium, plates were incubated at 25-27°C for 48-72 h. Pale-yellow, domed circular, and convex colonies with entire margins of 1-2 mm in diameter on yeast extract-dextrose-calcium carbonate (YDC) agar medium were appeared. All the obtained bacterial isolates were subjected to the standard biochemical tests. All the bacterial isolates were positive for oxidative metabolism; levan production, catalase; hydrolysis of tween 80, casein, aesculin; as well as HR on tobacco leaves. All isolates were negative for oxidase, urease and pectolytic activity, while have a weak amylolytic activity.Furthermore, bacterial isolates growth only on 1% and 3% NaCl, and did not grow on 5% NaCl and 0.1% triphenyl tetrazolium chloride (TTC). According to the phenotypic characteristics, walnut strains were identified as Xanthomonas sp. The isolates were further identified using generic and specific primers for Xanthomonads. The generic primer Xc-lip-F2/X-ITS-R2 capable to amplify a 777 bp fragment in all isolates, and confirmed the bacterial isolates as Xanthomonas sp. Type strains of X. alfalfae subsp. alfalfae (ICMP 15807) were included as controls in all biochemical, pathogenicity and molecular tests. Phylogenetic analysis, based on six housekeeping genes (hrpB2, fusA, gapA, gltA, lacF, and lepA), showed that all isolates as X. alfalfae subsp. alfalfae. Pathogenicity of the bacterial isolates were evaluated on alfalfa plants (cv. Bonab). In pathogenicity tests, water-soaked spots were appeared on alfalfa leaves 10–12 days post inoculation. Subsequently, the spots became yellowish brown and surrounded by chlorotic halos. To our knowledge, this is the first report of bacterial spot of alfalfa caused by X. alfalfae subsp. alfalfae in Iran.