cDNA-AFLP approach was employed to analyze genes differential expression under cold stress using the three-five leaf stage leaves of Medicago ruthenica.The results showed that 549 differentially expressed transcript-derived fragments（TDFs） were identified by sixty-four primer combinations,and 43 up-regulated TDFs of them were sequenced and analyzed.Blastx-analysis and functional analysis indicated that 32 TDFs might participate in senescence and metabolism pathways,signal transduction,transcription regulation,stress responses etc,other 11 TDFs were showed homology to unknown and hypothetical function genes.The qRT-PCR results showed that it was accuately on the numerical value for expression of three different up-regulated TDFs were induced by low temperature stress.