This study focused on the production of bio-oil from alfalfa stein material. Two alfalfa maturity stages, harvested at the early bud and full flower stages of development, were examined to evaluate the impact of variation in cell wall polysaccharide and lignin content on pyrolysis oil yields, production efficiency, and bio-oil and char quality, in terms of their use as combustion fuel and for chemicals. Findings included a lower-than-average yield of bio-oil and a higher-than-average yield of charcoal from alfalfa stems, compared to previous results for other biomass feedstocks. The bio-oil showed a decrease in oxygen content from the alfalfa stems, leading to a higher-than-average energy content in the bio-oil. Bio-oil yields were slightly higher for the more-mature alfalfa, which had higher levels of cell wall cellulose and lignin. Overall, when all the pyrolysis products were considered, energy recovery was better for the more-mature alfalfa stems.