Identifying quantitative trait loci (QTL) associated with alfalfa (Medicago sativa L.) shoot biomass (SB), water use efficiency, and C and N metabolism can resolve potential target sites for selection and provide new insight into genetic relationships that may exist between key traits in water-limited environments. The goal of this study was to utilize a tetraploid (2n = 4x = 32) BC1 mapping population and a genetic map based on 334 molecular markers to detect QTL associated with SB, C isotope discrimination (CID), and C and N content during drought stress. Half-sib families derived from 96 BC1 individuals were evaluated for the SB trait in seeded plots near Las Cruces, NM, during two water-stressed forage regrowth cycles in each of 3 yr (2005-2007). High costs associated with CID phenotyping precluded evaluation of the entire population. Therefore, in 2006 and 2007, shoot tissue samples for CID, C content, and N content measurements were collected from a subset of 29 families that exhibited extreme SB phenotypes in 2005. Significant genotype effects were observed among these families for SB and CID in both years and C and N content in 2006. Interval mapping identified QTL for all traits. Phenotypic effects of QTL supported significant positive genetic correlations detected between SB and CID, and between C and N content. Estimated QTL positions within the M. truncatula Gaertn. genome sequence assembly encompassed multiple candidate genes affiliated with stomatal development, cuticular wax biosynthesis, C and N metabolism, and abiotic stress response. [ABSTRACT FROM AUTHOR]