Gas exchange, water-use efficiency (WUE), and carbon-isotope discrimination (Δ) were compared among 14 populations of western larch (Larixoccidentalis Nutt.), a deciduous conifer. Seedlings grown from seed collected across the natural distribution of the species were maintained under water-stressed and well-watered conditions. No differences in gas exchange or instantaneous water-use efficiency (WUEi) were detected among populations. However, population differences in Δ, a second measure of water-use efficiency, were observed. WUE measured as the rate of dry matter accumulation per cumulative water transpired (WUET) was different among populations at the P = 0.07 level. The three measures of WUE were intercorrelated and all were higher among water-stressed seedlings than among well-watered seedlings. Overall means were 4.30 ± 0.08 (± SE) and 3.61 ± 0.07 g dry matter accumulation per kg water transpired (WUET), 2.80 ± 0.09 and 2.02 ± 0.06 μmol CO2 uptake per μmol H2O transpired (WUEi), and Δ was 18.24 ± 0.08‰ and 20.26 ± 0.10‰ under the water-stressed and well-watered treatments, respectively. Population differences in the two integrated measurements, WUET and Δ, were significantly correlated with the altitude at which seed was collected. No interaction between genotype and watering treatment was observed, which means that despite pronounced shifts in WUE between watering treatments, population rankings remained unchanged. These results provide evidence of genotypic differences in long-term measures of WUE among native populations of western larch.