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To obtain alfalfa genotypes with resistance to Verticillium wilt, in vitro selection using culture filtrate was conducted and the availability of this method was discussed. 20 regenerated plants were obtained from culture tissue which was derived from cotyledons of alfalfa cv. Kitawakaba (susceptible to Verticillium wilt) and selected for resistance to Verticillium albo-atrum culture filtrate. Of these, only 9 plants (45%) showed resistance to V. albo-atrum culture filtrate. Furthermore, 4 out of the 9 culture filtrate-resistant plants were susceptible to V. albo-atrum conidia inoculation. On the other hand, 11 culture filtrate-resistant plants were obtained in regenerants derived from non-selected callus tissue. These plants showed stable resistance to both V. albo-atrum culture filtrates and the conidia.