<>Alfalfa is one of the most important forage crops in the world owing to high yield, high protein content, and high economic benefits. Alfalfa is also a potential biofuel crop with advantage of low input cost. The complicated genetic inheritance in cultivated tetraploid alfalfa limits the power of QTL mapping and genetic investigation of important agronomic traits. In this study, we try to identify QTL related to biomass, biomass-related morphological traits, and biofuel related traits in two diploid F1 populations, one derived from intra-subspecies cross and the other from inter-subspecies cross. The frame genetic maps have been constructed using SSR markers. We are going to add more SNPs from biofuel-related candidate genes. Phenotypic data from different environments (growth chamber and field) are being collected. This study will provide new insight into improving the biomass yield of alfalfa in the breeding program.