Metal tolerance proteins (MTPs) encompass plant membrane divalent cation transporters to specifically participate in heavy metal stress resistance and mineral acquisition. However, the molecular behaviors and biological functions of this family inMedicago truncatulaare scarcely known. A total of 12 potentialMTPcandidate genes in theM. truncatulagenome were successfully identified and analyzed for a phylogenetic relationship, chromosomal distributions, gene structures, docking analysis, gene ontology, and previous gene expression.M. truncatula MTPs(MtMTPs) were further classified into three major cation diffusion facilitator (CDFs) groups: Mn-CDFs, Zn-CDFs, and Fe/Zn-CDFs. The structural analysis ofMtMTPsdisplayed high gene similarity within the same group where all of them have cation_efflux domain or ZT_dimer.Cis-acting element analysis suggested that various abiotic stresses and phytohormones could induce the mostMtMTPgene transcripts. Among allMTPs, PF16916 is the specific domain, whereas GLY, ILE, LEU, MET, ALA, SER, THR, VAL, ASN, and PHE amino acids were predicted to be the binding residues in the ligand-binding site of all these proteins. RNA-seq and gene ontology analysis revealed the significant role ofMTPgenes in the growth and development ofM. truncatula.MtMTPgenes displayed differential responses in plant leaves, stems, and roots under five divalent heavy metals (Cd2+, Co2+, Mn2+, Zn2+, and Fe2+). Ten, seven, and nineMtMTPsresponded to at least one metal ion treatment in the leaves, stems, and roots, respectively. Additionally,MtMTP1.1,MtMTP1.2, andMtMTP4exhibited the highest expression responses in most heavy metal treatments. Our results presented a standpoint on the evolution ofMTPsinM. truncatula. Overall, our study provides a novel insight into the evolution of theMTPgene family inM. truncatulaand paves the way for additional functional characterization of this gene family.