Alfalfa (Medicago sativa L.) possesses several attributes that makes it a key element for sustainable bio-ethanol production. It is a low input perennial legume widely adapted to very diverse environmental conditions with essentially no nitrogen fertilizer requirements. However research is needed to develop biofuel-type alfalfa with enhanced biomass production and standability, increased persistence, and superior cell wall degradability. In addition, genetic improvement for the accumulation of readily fermentable non-structural carbohydrates (NSC) could significantly increase the ethanol conversion rates from alfalfa biomass. Genetic gains for these traits are tributary to the availability of screening techniques for the precise identification of superior genotypes within populations with large genetic diversity. We recently undertook a screening project to identify genotypes with superior cell wall degradability and NSC accumulation within 300 genotypes randomly selected within six genetic backgrounds from European and North American origins. Biochemical analyses of dried stems revealed a large genetic variability for NSC content with concentrations ranging from 20 to 100 mg g^-1 DW. Interestingly, NSC variability was markedly higher in a genetic background of European origin as compared to the other populations, further highlighting the potential for genetic improvement for that trait. A modified commercial enzymatic cocktail (Accelerase^TM 1000, Genencor) is currently being developed to optimize the degradation of alfalfa biomass.  An efficient enzymatic assay specifically designed for alfalfa biomass is a prerequisite to the identification of genotypes with contrasted degradability. Moreover, DNA extracted from genotypes with superior (30 highest) and inferior (30 lowest) cell wall degradability and/or NSC accumulation will be pooled and used for bulk segregant analysis of DNA polymorphisms using the PCR-based sequence-related amplified polymorphism technique. Identification of genetic markers associated with these traits will help accelerate the commercial release of biofuel-type cultivars of alfalfa.