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We examined genetic differentiation of intrinsic water-use efficiency (WUE) and the relationship between WUE and phyllode morphological and nutritional traits in the native Hawaiian tree koa (Acacia koa Gray, Fabaceae). We obtained both integrated (stable isotope C-13 composition of phyllode tissues; delta C-13) and instantaneous (assimilation/stomatal conductance to water vapor; A/g) estimates of WUE on koa accessions grown in a progeny trial at Maunawili on the island of O’ahu. The accessions were from sites along an elevation (900-1300 m) and rainfall (ca. 2200-1300 mm) gradient on the southwestern slopes of Mauna Loa on the island of Hawai’i. The delta C-13 values of koa phyllodes at Maunawili ranged between -28 parts per thousand and -31 parts per thousand and were positively related to elevation of the seed source. Among seed sources grown at the common site, phyllode delta C-13 varied with similar magnitude and direction to that associated with the field populations, indicating genetic differentiation of WUE in koa. Instantaneous measurements of WUE showed similar trends of increasing WUE with elevation of the seed source. Phyllode delta C-13 was not correlated with foliar concentrations of nitrogen (N), indicating that differences in delta C-13 were not caused by nutrient-related changes in photosynthetic capacity but rather by stomatal limitation. Phyllode morphological traits such as specific leaf mass (SLM), length, and area were unrelated to delta C-13. The similarity between the slopes of SLM and phyllode N content at Maunawili and Honaunau supported the contention that there is global interdependence between these two traits. The finding of genetic differentiation of WUE in koa is important for native forest restoration and seed selection.