Dehydration-responsive element-binding proteins (DREBs) belong to members of the AP2/ERF transcription factor superfamily, which has been reported to involve various abiotic-stress responses and tolerance in plants. However, research on the DREB-family is still limited in alfalfa (Medicago sativa L.), a forage legume cultivated worldwide. The recent genome-sequence release of the alfalfa cultivar “XinJiangDaYe” allowed us to identify 172 DREBs by a multi-step homolog search. The phylogenetic analysis indicated that such MsDREBs could be classified into 5 groups, namely A-1 (56 members), A-2 (39), A-3 (3), A-4 (61) and 13 (A-5 (13), thus adding substantial new members to the DREB-family in alfalfa. Furthermore, a comprehensive survey in silico of conserved motif, gene structure, molecular weight, and isoelectric point (pI) as well as gene expression was conducted. The resulting data showed that, for cold-stress response, 33 differentially expressed MsDREBs were identified with a threshold of Log2-fold > 1, and most of which were transcriptionally upregulated within 48 h during a cold treatment(s). Moreover, the expression profiling of MsDREBs from two ecotypes of alfalfa subspecies i.e. M. sativa ssp. falcata (F56, from a colder region of Central Asia) and M. sativa ssp. sativa (B47, from Near East) revealed that most of the cold-stress responsive MsDREBs exhibited a significantly lower expression in F56, leading to a proposal of the existence of a distinct mechanism(s) for cold tolerance regulated by DREB-related action, which would have been evolved in alfalfa with a genotypic specificity. Additionally, by examining the transcriptome of a freezing-tolerance species (M. sativa cv. Zhaodong), eight DREBs were found to be implicated in a long-term freezing-stress adaptation with a great potential. Taken together, the current genome-wide identification in alfalfa points to the importance of some MsDREBs in the cold-stress response, providing some promising molecular targets to be functionally characterized for the improvement of cold tolerance in crops including alfalfa.