Abstract: In order to isolate the high-temperature stress-induced ESTs from alfalfa, we took the alfalfa variety‘ABI 700’as material whose fall dormancy level was 6, and taking the cDNA from alfalfa tender stems and leaves under 45℃ as tester, while taking their cDNA under 22℃ (normal growth) as driver. The forward suppression subtractive library of alfalfa under high temperature stress was constructed by using suppression subtractive hybridization. Choosing 120 positive clones stochastically from the subtractive library to take bacterial PCR, it displayed the recombination rate of library clones was 97.8%. The size of inserted fragment was mainly between 300-750 bp. This research of the alfalfa differential expression has laid a theoretical basis for the cloning of heat-resistance gene and the gene expression under high temperature.