Heterosis is an effective way to increase yield and improve quality in alfalfa breeding. The key issue for efficiently use of heterosis is to identify parents with potential to produce hybrid combinations with higher performance. This study aimed to analyze the genetic distance between the 45 alfalfa accessions, and combine different hybrid combinations. The results showed that the mating ability of hybrid combinations in which both parents are tetraploid is greater than that in which both parents are diploid or in hybrid combination with different parental ploidy. In terms of compatibility, combinations were ranked from largest to smallest as follows: diploidsｘtetraploids, tetraploidsｘtetraploids, diploidsｘdiploids, tetraploidsｘdiploids. For parents of the same ploidy level analysis, the mating ability of the hybrid combinations in which both parents were tetraploid was greater than those in which both parents were diploid. For parents of the different ploidy level analysis, the mating ability of alfalfa hybrid combinations with a diploid as female parent was better than those of alfalfa hybrid combinations with a tetraploid as female parent. Moreover, CYK2ｘHH10 with high heterosis and CYK4xHH10 with high relative seed setting rate should be used as candidate materials for further investigation.