The goal of this project is to identify genetic mechanisms associated with drought tolerance in tetraploid alfalfa (Medicago sativa L). Two first generation backcross mapping populations (~100 genotypes each) were generated between a high yielding, low water use efficient parent (M. sativa subsp. sativa var. ‘Chilean') and a low yielding, high water use efficient parent (M. sativa subsp. falcata var ‘Wisfal'). Half-sib seed was produced from each mapping population genotype and seeded into replicated field plots at Las Cruces, NM in fall 2004. Forage biomass was measured in two harvests each in 2005 and 2006 that received 50% of the normal irrigation rate to induce episodic drought stress. Significant variation among families was detected in both populations, and some families performed similarly to the drought tolerant check, ‘Wilson'. Evaluation of 2005 yield data, in conjunction with 286 EST-SSR markers, identified individual alleles that accounted for up to 15% of yield variance under drought conditions. The magnitude of marker allele effects differed between harvests, but the direction of their influence was relatively consistent. The 2006 yield data verified the influence of several of these loci. Potential epistatic interactions between unlinked markers were also revealed. Additional markers derived from drought responsive genes possessing single nucleotide polymorphisms are also being mapped and evaluated for their associations with yield under drought stress.