Fertile kanamycin resistant plants of Medicago sativa L. were obtained by genetic transformation with binary vector of Agrobacterium tumefaciens and were used as a fertilizer of Lethionine (ETH) resistant alfalfa variants. The dominant nature of both kanamycin and Lethionine resistant traits was demonstrated by the double resistant phenotype of obtained hybrid plants. Southern blot analysis showed that transgenic plants and self-pollinated progeny harbored the kanamycin gene, and expressed the neomycin phosphotransferase (npt) activity in vitro. The inheritance of both single (kanamycin) and double (kanamycin and Lethionine) resistance traits was analyzed and the way in which marker characters are segregated is discussed.