Summary• Whole-genome multiplication, or polyploidy, is common in angiosperms and many species consist of multiple cytotypes that have different physiological tolerances. However, the relative importance of genome duplication vs post-duplication evolutionary change in causing differentiation between cytotypes is not known.• We examined the water relations of Chamerion angustifolium, a herbaceous perennial in which diploid and tetraploid cytotypes occupy different niches. To differentiate between the effects of genome duplication and evolutionary changes that followed polyploidization, we compared extant diploids and tetraploids with experimentally synthesized neotetraploids.• Tetraploids had 32% higher xylem hydraulic conductivity (K H ) than neotetraploids and 87% higher K H than diploids, but vulnerability to water stress induced cavitation and gas exchange sensitivity to water potential did not differ among cytotypes. Nevertheless, tetraploids took 22% and 30% longer to wilt than neotetraploids and diploids. A simple hydraulic model suggested that tetraploids deplete soil moisture to a greater degree than neotetraploids and diploids before reaching leaf water potentials that cause stomatal closure.• We conclude that the different physiological tolerances and distribution of diploid and tetraploid C. angustifolium are unlikely to be caused solely by genome duplication. The enhanced ability of tetraploids to survive water stress likely evolved after polyploidization.