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BACKGROUND Homeodomain leucine zipper (HD-ZIP) transcription factors play roles in regulating plant development and responses to abiotic stresses; however, how HD-ZIP genes in Medicago truncatula are involved in abiotic stress response remains elusive. METHODS AND RESULTS The HD-ZIP I genes in Medicago truncatula were identified and characterized, and their expression patterns in different tissues and under different abiotic stresses were analyzed. A total of 15 Medicago truncatula HD-ZIP I genes were identified and a phylogenetic analysis of HD-ZIP I proteins in Arabidopsis thaliana and Medicago truncatula was conducted. Fifteen HD-ZIP I genes showed diverse tissue preferences. Among them, expressions of MtHB22 and MtHB51 were specially detected in vegetative buds. In addition, they responded to various abiotic stresses, including salinity and osmotic stress and abscisic acid (ABA). For instance, MtHB7 and MtHB12 expression levels were found to be positively associated with salt, osmotic stress and ABA in both shoots and roots, while MtHB13 and MtHB23 were negatively associated with these stresses in Medicago truncatula. CONCLUSION The HD-ZIP I genes in Medicago truncatula are evolutionarily conserved, but also exhibit gene duplication and gene loss events. Differential expression analysis of Medicago truncatula HD-ZIP I genes indicated their crucial roles in abiotic stress responses. Our genome-wide analysis of the HD-ZIP I transcription factor family in Medicago truncatula provided a valuable reference for further research.