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Alfalfa is a worldwide grown forage crop and is important due to its high biomass production and nutritional value. However, the production of alfalfa is challenged by adverse environmental factors such as drought and other stresses. Developing drought resistance alfalfa is an important breeding target for enhancing alfalfa productivity in arid and semi-arid regions. In the present study, we used genotyping-by-sequencing and genome-wide association to identify marker loci associated with biomass yield under drought in the field in a panel of diverse germplasm of alfalfa. A total of 28 markers at 22 genetic loci were associated with yield under water deficit, whereas only four markers associated with the same trait under well-watered condition. Comparisons of marker-trait associations between water deficit and well-watered conditions showed non-similarity except one. Most of the markers were identical across harvest periods within the treatment, although different levels of significance were found among the three harvests. The loci associated with biomass yield under water deficit located throughout all chromosomes in the alfalfa genome agreed with previous reports. Our results suggest that biomass yield under drought is a complex quantitative trait with polygenic inheritance and may involve a different mechanism compared to that of non-stress. BLAST searches of the flanking sequences of the associated loci against DNA databases revealed several stress-responsive genes linked to the drought resistance loci, including leucine-rich repeat receptor-like kinase, B3 DNA-binding domain protein, translation initiation factor IF2, and phospholipase-like protein. With further investigation, those markers closely linked to drought resistance can be used for MAS to accelerate the development of new alfalfa cultivars with improved resistance to drought and other abiotic stresses.