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ABSTRACT Sampling of stable isotopes in plant xylem water (δ²H, δ¹⁸O) has become a ubiquitous technique to study spatiotemporal variations in the water taken up by plant roots; however, open questions remain concerning the most appropriate time of day to sample trees to obtain representative xylem water isotopic values (δ_XYLEM). We sampled the δ_XYLEMof oak and maple trees prior to solar midday (i.e., in a recommended sampling window) and then again after solar midday (i.e., outside of the recommended window) across 4 months. The paired root mean squared difference between AM and PM δ¹⁸O ranged from 1.00‰ to 1.16‰ for maples and 0.23‰ to 2.55‰ for oaks across all sampling dates. Xylem water seasonal origin index (SOI) values derived from AM and PM δ_XYLEMsamples were significantly different, though both SOI estimates supported the conclusion that maple and oak δ_XYLEMreflected summer precipitation on all sampling dates. We conclude that sampling time of day is a significant consideration in the design of δ_XYLEMsampling campaigns; however, our findings also support flexibility in the collection time of δ_XYLEMin field sites where sampling during the optimal time of day is challenging.