Carbon isotope discrimination (Delta) has been proposed as a method for evaluating transpiration efficiency in C-3 plants. It is important to study the association of A in different parts with the long-term water use efficiency (WUE). In this paper, plant height, Delta in leaf water soluble carbohydrates (Delta(WSC)), photosynthetic gas exchange parameters, Delta in aboveground shoot (Delta(AGS)), stem/leaf weight ratio, aboveground biomass (AGB), evapotranspiration, and WUE were determined in a collection of 10 alfalfa (Medicago sativa L.) genotypes under three water treatments (230 [T-1], 460 [T-2], and 700 mm irrigation [T-3]) in a Ningxia central desert steppe in 2012 and 2013. Significant interactions were found for AGB and WUE among year, treatment, and genotype, whereas significant interactions of year x treatment and year x genotype were only found for Delta(AGS) but not for Delta(WSC). Generally, more water input increased the values of the traits tested except for WUE and single leaf intrinsic WUE (P-n/G(s)). More irrigated water input resulted in increase in AGB, Delta(AGS), and Delta(WSC) and decrease in P-n/G(s). The greatest WUE was recorded under T-2 in 2012. Significant and positive correlations were observed between AGB and Delta(AGS) (r = 0.588, 0.574, and 0.386 under T-1, T-2, and T-3, respectively). Under T-2 and T-3, A(WSC) correlated negatively with WUE (r = -0.383 and 0.602, respectively). Ningmu No. 1 was found to have the greatest WUE (18.34 kg ha(-1) mm(-1)) and yield (17.71 Mg ha(-1)) across water treatments and years. The findings suggest that in the semiarid areas applying irrigation of 230 to 700 mm yr(-1), Delta in aboveground shoots or in leaf water-soluble carbohydrates at the flowering stage seems to be a useful indicators reflecting aboveground biomass and integrated WUE for alfalfa.