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The increasing demand for food production and predicted climate change scenarios highlight the need for improvements in crop sustainability. The efficient use of water will become increasingly important for rain-fed agricultural crops even in fertile regions that have historically received ample precipitation. Improvements in water-use efficiency in Zea mays have been limited, and warrant a renewed effort aided by molecular breeding approaches. Progress has been constrained by the difficulty of measuring water-use in a field environment. The stable carbon isotope composition (delta C-13) of the leaf has been proposed as an integrated signature of carbon fixation with a link to stomatal conductance. However, additional factors affecting leaf delta C-13 exist, and a limited number of studies have explored this trait in Z. mays. Here we present an extensive characterization of leaf delta C-13 in Z. mays. Significant variation in leaf delta C-13 exists across diverse lines of Z. mays, which we show to be heritable across several environments. Furthermore, we examine temporal and spatial variation in leaf delta C-13 to determine the optimum sampling time to maximize the use of leaf delta C-13 as a trait. Finally, our results demonstrate the relationship between transpiration and leaf delta C-13 in the field and the greenhouse. Decreasing transpiration and soil moisture are associated with decreasing leaf delta C-13. Taken together these results outline a strategy for using leaf delta C-13 and reveal its usefulness as a measure of transpiration efficiency under well-watered conditions rather than a predictor of performance under drought.