Abstract Background Freezing provokes severe yield losses to different fall-sown annual legumes. Understanding the molecular bases of freezing tolerance is of great interest for breeding programs. Medicago truncatula Gaertn. is an annual temperate forage legume that has been chosen as a model species for agronomically and economically important legume crops. The present study aimed to identify positional candidate genes for a major freezing tolerance quantitative trait locus that was previously mapped to M. truncatula chromosome 6 (Mt-FTQTL6) using the LR3 population derived from a cross between the freezing-tolerant accession F83005-5 and the freezing-sensitive accession DZA045-5. Results The confidence interval of Mt-FTQTL6 was narrowed down to the region comprised between markers MTIC153 and NT6054 using recombinant F 7 and F 8 lines. A bacterial-artificial chromosome (BAC) clone contig map was constructed in an attempt to close the residual assembly gap existing therein. Twenty positional candidate genes including twelve C-repeat binding factor (CBF)/dehydration-responsive element binding factor 1 (DREB1) genes were identified from BAC-derived sequences and whole-genome shotgun sequences (WGS). CBF / DREB1 genes are organized in a tandem array within an approximately 296-Kb region. Eleven CBF / DREB1 genes were isolated and sequenced from F83005-5 and DZA045-5 which revealed high polymorphism among these accessions. Unique features characterizing CBF / DREB1 genes from M. truncatula , such as alternative splicing and large tandem duplication, are elucidated for the first time. Conclusions Overall, twenty genes were identified as potential candidates to explain Mt-FTQTL6 effect. Their future functional characterization will uncover the gene(s) involved in freezing tolerance difference observed between F83005-5 and DZA045-5. Knowledge transfer for breeding improvement of crop legumes is expected. Furthermore, CBF / DREB1 related data will certainly have a large impact on research studies targeting this group of transcriptional activators in M. truncatula and other legume species.