Heterotic grouping of 40 Alfalfa materials(six male sterile lines and 34 alfalfa breeding materials) genetic distance were analyzed by SSR markers, and using the six sterility strain and 14 alfalfa varieties test cross lines, in further to analysis for genetic distance (GD), yield combining ability and heterosis effect. The results show that, article 25 for SSR primers amplification out 189 band, including 136 polymorphic bands, the average polymorphism loci percentage was 69.23%. The genetic distance of 40 copies of alfalfa materials was 0.1818-0.9091, an average of 0.4544. Each parent effect of general combining ability were significant positive correlation with alfalfa heterosis, and GD ware also that, the correlation between general combining ability and heterosis was higher than GD and heterosis. Therefore, SSR genetic distance was not enough to assemble strong hetrosis combination, should be based on the character analysis of combining ability.