Photosynthesis (A), water relations and stomatal reactivity during drought, and leaf morphology were evaluated on 2-year-old, sun- and shade-grown Prunus serotina Ehrh. seedlings of a mesic Pennsylvania seed source and a more xeric Wisconsin source. Wisconsin plants maintained higher A and leaf conductance (gwv) than Pennsylvania plants during the entire drought under sun conditions, and during the mid stages of drought under shade conditions. Compared to shade plants, sun plants of both sources exhibited a more rapid decrease in A or % Amax with decreasing leaf water potential (Ψ). Tissue water relations parameters were generally not significantly different between seed sources. However, osmotic potentials were lower in sun than shade plants under well-watered conditions. Following drought, shade plants, but not sun plants, exhibited significant osmotic adjustment. Sun leaves had greater thickness, specific mass, area and stomatal density and lower guard cell length than shade leaves in one or both sources. Wisconsin sun leaves were seemingly more xerophytic with greater thickness, specific mass, and guard cell length than Pennsylvania sun leaves. No source differences in leaf structure were exhibited in shade plants. Stomatal reactivity to sun–shade cycles was similar between ecotypes. However, well-watered and droughted plants differed in stomatal reactivity within and between multiple sun–shade cycles. The observed ecotypic and phenotypic variations in ecophysiology and morphology are consistent with the ability of Prunus serotina to survive in greatly contrasting environments.