Alfalfa (Medicago sativa L.) is a perennial legume that is widely cultivated as a forage crop for its high yield and forage quality, perennial growth habit, and adaptability to different environmental conditions. Alfalfa is a photoperiod sensitive long-day plant species. Flowering time is an important agronomic trait for crops in general. In alfalfa, the onset of flowering is negatively associated with biomass yield and nutritional quality due to increased lignin accumulation in stems and nutrient recycling from leaves to support seed development. Late flowering varieties are advantageous for forage production as the extended growth time enables more vegetative growth and forage production. In forage crop production, high dry matter, digestibility, and other nutritional traits are strongly affected by flowering. Consequently, late flowering for biomass yield and quality enhancement become a desirable breeding need in forage crop production. We attempted to delay flowering in alfalfa using an optimized multiplex CRISPR/Cas9-mediated mutagenesis of the key flowering time master regulator gene FLOWERING LOCUS Ta1 (MsFTa1). Four guide RNAs in different exons of MsFTa1 were arranged in a polycistronic tRNA-gRNA system and introduced into alfalfa by Agrobacterium-mediated transformation. Ninety-six putative mutant lines were identified by sequencing and characterized for flowering time and desirable agronomic traits. Phenotype assessment of flowering time identified 22 independent mutant lines with delayed flowering time up to 21 days. Six homozygous Msfta1 mutant lines containing mutations in all four copies of MsFTa1 showed forage biomass with increases of up to 78% in fresh weight and 76% in dry weight compared to controls. Depending upon the harvesting schemes, many of these lines also had reduced lignin, ADF and NDF content but significantly higher crude protein content. These CRISPR/Cas9-edited Msfta1 mutant lines could be introduced into alfalfa breeding programs to generate elite transgene-free alfalfa cultivars with improved forage biomass yield and quality.