The development of deuterated biomass is essential for effective neutron scattering studies on biomass, which can provide key insights into the complex biomass conversion processes. A method for optimized production of deuterated annual ryegrass (Lolium multiflorum) was developed by growing the plants in 50% D2O in perfused hydroponic chambers. Deuterium incorporation of 36.9% was found in the annual rye grown in 50% D2O. Further, deuterium incorporation of 60% was achieved by germinating the rye seedlings in H2O and growing in 50% D2O inside the perfusion chambers. The characteristics related to enzymatic hydrolysis such as biomass composition, degree of polymerization, and cellulose crystallinity were compared with its control protiated counterpart. The cellulose molecular weight indicated slight variation while hemicellulose molecular weights and cellulose crystallinity remain unaffected with the deuteration.