The buds of male sterile line of alfalfa MS4 and its maintainer were taken as materials, employed the suppression subtractive hybridization (SSH) technology. Two differential gene expression subtractive cDNA libraries were constructed under sterile and fertile conditions. Positive clones were randomly selected to sequence and analyze by Blast. 190 ESTs with known functions were got. 6 genes were inferred which may be associated with male fertility of alfalfa by GO functional classification. The expression of 6 genes were analyzed by fluorescence quantitative PCR, the results showed 6 genes had differential expression at different development stages in the male sterile line of alfalfa. It suggested that these 6 genes may be related to the male fertility of alfalfa, and play an important role in fertility transformation.