VQ motif-containing proteins play important roles in plant abiotic and biotic stresses.In this study, VQ gene family were identified and systematically analyzed from Medicago sativa. A total of 24 MsVQ genes were identified in the alfalfa genome, which were unevenly distributed on eight chromosomes. Expect from MsVQ8 and MsVQ17, MsVQ genes do not contain introns and a series of MsVQs were predicted to localized at nucleus. Phylogenetic analysis showed that MsVQ was divided into seven subgroups, each subfamily shared relatively conserved gene structures and protein motifs. Medicago sativa genome included 6 pairs of segmental duplicated genes and 1 pair of tandem repeat genes. The abiotic stress-responsive elements, hormone-responsive elements and WRKY binding box were presented in the promoters of MsVQs. Transcriptome data showed that MsVQs were respond to abiotic stresses such as drought, salt and low temperature. The quantitative real-time PCR (qRT-PCR) found that three MsVQs genes were significantly induced by salt stress and six MsVQs genes expression levels reached a peak at 6h after alkaline stress. Taken together, these findings will provide clues for further study of MsVQs functions and alfalfa molecular breeding.