Technical Abstract: Alfalfa (Medicago sativa L.) has potential utility as an energy crop for conversion to biofuels because it is already produced commercially, grows as a perennial, increases soil nitrogen, and the protein enriched leaves can be marketed as a co-product for animal feed. In this paper, the biomass processing characteristics of the stem material were evaluated for biochemical conversion into ethanol. To evaluate the potential of plant cell wall engineering to enhance product yields, a reduced S-lignin transgenic genotype generated through down regulation of the caffeic acid O-methyltransferase (COMT) gene was compared to its wild-type counterpart. Early and late cuttings were examined for chemical compositions. The samples had similar carbohydrate contents including a mean composition of 316 g glucan and 497 g total neutral carbohydrates per kg dry biomass, which corresponds to an average theoretical ethanol yield of 299 l/ton. Samples were pretreated with dilute-acid or dilute ammonium hydroxide and the whole hydrolysates evaluated for enzymatic and ethanol conversion efficiencies. The results varied with pretreatment. When pretreated with acid, enzymatic and ethanol conversion efficiencies were improved for late vs. early harvested samples. The only significant difference associated with the COMT genotype was for ethanol conversion, where it was associated with an 8% improvement. In contrast when treated with alkali, the COMT genotype displayed both enhanced enzymatic and fermentation conversion efficiencies. The COMT genotype was associated with significantly higher glucose and ethanol conversion efficiencies. When converted using a recombinant xylose-fermenting Saccharomyces strain, the mean ethanol conversion efficiency was also improved by 8%. Ethanol yields per ton biomass were 204–241 liters.