Technical Abstract: To have a comprehensive understanding of how legume plants respond to drought at the gene expression level and examine whether legume plants that are not fixing nitrogen would behave similar to non-legume plants in drought response, transcriptomes were studied in two non-nodulated alfalfa (Medicago sativa L.) cultivars, Ladak and 53V08, when plants were subjected to dehydration stress. Two heat shock-related protein genes were up-regulated in the 3-h stressed shoots in both cultivars. One of them was also up-regulated in the 8-h stressed shoots, along with dehydrin and LEA. A xyloglucan endotransglycosylase and a gene with unknown function were down-regulated in both 3- and 8-h stressed shoots. In roots, nearly half of the 55 genes commonly up-regulated at 3 h are involved in pathogen resistance, insect defense and flavonoid synthesis, which differs from other dehydration - responsive genes, such as LEA and dehydrin, were up-regulated after 8 h of treatment. The genes encoding caffeoyl-CoA O-methyl transferase and dirigent were up-regulated in the 3-h stressed roots, while two aquaporin genes were down-regulated, suggesting that lignification and prevention of water loss in roots in initial dehydration stress is a common strategy for both cultivars. The results also indicate the involvement of some specific signal transduction pathways, osmotic adjustment and ion homoeostasis regulation during dehydration response. Besides those known dehydration - responsive genes in the literature, some dehydration responses and genes in alfalfa appear to be unique. Our results provide valuable insight into a comprehensive understanding of dehydration response in alfalfa at the molecular level. Electronic supplementary material: The online version of this article (doi:10.1007/s11738-007-0l07.5) contains supplementary material, which is available to authorized users.