TheDoftranscription factor is a plant-specific transcription gene family that plays various biological functions in plant development and stress response. However, no relevant research has been conducted onMedicago polymorpha. Here, 36MpDofgenes were identified in theM. polymorphagenome and further divided into 10 groups based on the comparative phylogenetic analysis. The essential information ofMpDofgenes, such as chromosomal localization, gene structure, conserved motifs, and selective pressures were systematically analyzed. All 36MpDofgenes were predicted to contain morecis-acting elements related to hormone response.MpDof24andMpDof25were predicted to interact withMpDof11andMpDof26to involve in the photoperiod blooms process. TheMpDofgenes showed a diverse expression pattern in different tissues. Notably,MpDof29andMpDof31were specifically expressed in the large pod and root, respectively, suggesting their crucial role in the pod and root development. qRT-PCR analysis indicated that the expression levels ofMpDof10,MpDof25,MpDof26, andMpDof29were obviously up-regulated under drought, salt, and cold stress. Collectively, genome-wide identification, evolutionary, and expression analysis of theDoftranscription gene family inM. polymorphawill provide new information to further understand and utilize the function of theseDofgenes inMedicagoplants.