BackgroundNIN-like protein (NLP) transcription factors (TFs) compose a plant-specific gene family whose members play vital roles in plant physiological processes, especially in the regulation of plant growth and the response to nitrate-nitrogen. However, no systematic identification or analysis of the NLP gene family has been reported in alfalfa. The recently completed whole-genome sequence of alfalfa has allowed us to investigate genome-wide characteristics and expression profiles.Results53MsNLPgenes were identified from alfalfa and renamed according to their respective chromosome distributions. Phylogenetic analysis demonstrated that theseMsNLPscan be classified into three groups on the basis of their conserved domains. Gene structure and protein motif analyses showed that closely clusteredMsNLPgenes were relatively conserved within each subgroup. Synteny analysis revealed four fragment duplication events ofMsNLPsin alfalfa. The ratios of nonsynonymous (Ka) and synonymous (Ks) substitution rates of gene pairs indicated that theMsNLPgenes underwent purifying selection during evolution. Examination of the expression patterns of different tissues revealed specific expression patterns of theMsNLPgenes in the leaves, indicating that these genes are involved in plant functional development. Prediction of cis-acting regulatory elements and expression profiles further demonstrated that theMsNLPgenes might play important roles in the response to abiotic stress and in phytohormone signal transduction processes.ConclusionThis study represents the first genome-wide characterization ofMsNLPin alfalfa. MostMsNLPsare expressed mainly in leaves and respond positively to abiotic stresses and hormonal treatments. These results provide a valuable resource for an improved understanding of the characteristics and biological roles of theMsNLPgenes in alfalfa.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-023-09418-x.