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Calcium is an important second messenger in mediating adaptation responses of plants to abiotic and biotic stresses. Calmodulin-like (CML) protein is an important calcium-signaling protein that can sense and decode Ca²⁺ signal in plants. <i>Medicago truncatula</i> is a model legume plant; however, investigations of MtCML proteins are limited. Using genome analysis and BLAST database searches, fifty MtCML proteins that possess EF-hand motifs were identified. Phylogenetic analysis showed that CML homologs between <i>M. truncatula</i>, <i>Arabidopsis thaliana</i> and <i>Oryza sativa</i> shared close relationships. Gene structure analysis revealed that these <i>MtCML</i> genes contained one to four conserved EF-hand motifs. All <i>MtCMLs</i> are localized to eight chromosomes and underwent gene duplication. In addition, <i>MtCML</i> genes were differentially expressed in different tissues of <i>M. truncatula</i>. <i>Cis</i>-acting elements in promoter region and expression analysis revealed the potential response of MtCML protein to abiotic stress and hormones. The results provide a basis of further functional research on the <i>MtCML</i> gene family and facilitate their potential use for applications in the genetic improvement on <i>M. truncatula</i> in drought, cold and salt stress environments.