The wall-associated kinases (WAKs) can perceive and transmit extracellular signals as one kind of unique receptor-like kinases (RLKs) involved in the regulation of cell expansion, pathogen resistance and abiotic stress tolerance. To understand their potential roles and screen some key candidates inMedicago truncatula(M. truncatula), genome-wide identification and characterization ofMtWAKswere conducted in this study. A total of 54MtWAKgenes were identified and classified into four groups based on their protein domains. They were distributed on all chromosomes, while most of them were clustered on chromosome 1 and 3. The synteny analysis showed that 11 orthologous pairs were identified betweenM. truncatulaandArabidopsis thaliana(A.thaliana) and 31 pairs betweenM. truncatulaandGlycine max(G.max). The phylogenetic analysis showed that WAK-RLKs were classified into five clades, and they exhibited a species-specific expansion. MostMtWAK-RLKshad similar exon–intron organization and motif distribution. Multiplecis-acting elements responsive to phytohormones, stresses, growth and development were observed in the promoter regions ofMtWAK-RLKs. In addition, the expression patterns ofMtWAK-RLKsvaried with different plant tissues, developmental stages and biotic and abiotic stresses. Interestingly, plasm membrane localized MtWAK24 significantly inhibitedPhytophthorainfection in tobacco. The study provides valuable information for characterizing the molecular functions ofMtWAKsin regulation of plant growth, development and stress tolerance in legume plants.