Carbon isotope discrimination (Δ) has been proposed as a method for evaluating water use efficiency (WUE) in C3 plants and as a precise technique for screening plants with higer tolerance under water deficit conditions. In this research, 18 alfalfa genotypes from different geographical origins were evaluated under irrigated and rain-fed conditions in organically managed fields in Austria. Significant differences were found amongst harvests for Δ-shoot under both conditions while genotype by harvest interaction was only significant under irrigated condition. Drought stress under rain-fed condition reduced the overall mean of water use efficiency and carbon isotope discrimination responses(up to 34%), but the ratios of reduction differed for characters and genotypes. Narrow ranges were found for all traits especially for WUE-TBY (total biomass yield) (0.78 kg m−3) and Δ-shoot (0.53‰) based on genotype means over locations and years, although variation and ranges were higher under irrigated condition. Regarding the variable and low correlations, simultaneous assessment of genotypes for Δ-shoot and biomass production can ensure the selection of superior genotypes and minimize potential biomass reductions that may result from using Δ-shoot as the only selection criterion to improve WUE. Sitel was the most water use efficient genotype(2.79 and 4.48 kg m−3 based on shoot dry matter and total biomass,respectively) across two condition (widely adapted genotype) followed by Mohajeran, Fix232 and Verko under irrigated condition (as specific adapted genotypes) and Vlasta, Sanditi, Ghara-aghaj under rain-fed condition.