Plant improvement to increase the water‐use efficiency of affalfa (Medicago sativa L.) has been limited. This study compared leaf color variants (pale and dark) to evaluate the effect of alfalfa leaf chlorophyll on water relations and yield of alfalfa. Leaf color populations were selected from ‘Ladak 65’ and visually selected sub‐populations evaluated under controlled‐environment and field conditions. Seven moisture regimes were established in controlled environment studies in which a commercial root medium with high water‐holding capacity was used. The field study was at Laramie, WY, on a Wyocolo soil (fine, loamy, mixed Borollic Haplargid) and consisted of irrigated and nonirrigated treatments. Traits measured for the two populations included leaf chlorophyll (Chl), leaf photo‐synthesis (as CO2 exchange rate: CER), transpiration (T), stem pressure potential (ψw), water‐use efficiency (WUE), leaf area, forage yield. Controlled. environment conditions produced pale plants that contained 15% less total Chl than dark‐leaved variants. The Chl concentration of pale variants was equal between the moisture extremes, while the dark variants decreased 20% for the low‐moisture (compared with high moisture) regime in the controlled environment. The WUE in the controlled environment of pale and dark leaf types averaged 1.47 and 1.22 g dry forage kg−1 water, respectively. This difference in WUE was attributed mostly to the greater growth of pale leaf types. The CER and CER/T ratio of the dark leaf types were more sensitive to moisture stress than the pale types. In the 2‐yr field study, the pale leaf types had a 22% lower CER than the dark leaf types across moisture regimes and 19% lower T under the irrigated regime. Even though leaf photosynthesis was lower for the pale leaf types, the forage yield was 25% higher under the irrigated regime.

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