Elucidation of the mechanisms of dehydration tolerance in poplar (Populus sp.) trees will permit development of biochemical and molecular indicators to identify dehydration-tolerant genotypes during genetic selection. The objectives of this study were to characterize the degree of phenotypic variation in osmotic potential (a determinant of dehydration tolerance), determine the relationship between osmotic potential at full turgor and relative growth rate, and identify quantitative trait loci (QTL) for osmotic potential in an advanced-generation, interspecific poplar pedigree established in contrasting environments. A three-generation, sib-mated black cottonwood (Populus trichocarpa Torr. & Gray) and eastern cottonwood (P. deltoides Bartr.) segregating F(2) family (Family 331) was analyzed at a dry site east of the Cascade Mountain Range (Boardman, OR) and at a wet site west of the mountains (Clatskanie, OR). At the Boardman site, 2-year-old trees (59 clones) were either irrigated everyday (wet) or every other day (dry), whereas 3- and 4-year-old trees (58 clones) at the Clatskanie site were unirrigated. At the Boardman site, the typically narrow range of osmotic potentials exhibited by grandparents and parents was greatly expanded in the F(2) population, spanning from -1.38 to -2.35 MPa under wet conditions, with a similar range under dry conditions (-1.40 to -2.15 MPa). Clones that had osmotic potentials < or = -1.90 MPa generally displayed full maintenance of stem relative growth rates under dry conditions in contrast to clones with osmotic potentials that were < or = -1.60 MPa, in which stem relative growth rates were reduced by an average of 38% in the dry treatment relative to the wet treatment. Although osmotic adjustments of 0.13 to 0.36 MPa were observed in nine out of 59 clones, adjustment typically occurred from relatively high baseline osmotic potentials. The range in osmotic potential at the wetter Clatskanie site at age three was higher (-1.27 to -1.84 MPa) and was further expanded the following year (-1.14 to -1.94 MPa), which had a wetter spring than the previous year, followed by a typically dry July. Seven QTL for osmotic potential were identified that each explained > 7.5% of the variation in osmotic potential. Given that four clones (7%) had osmotic potentials of -2.00 MPa or less and that QTL for osmotic potential have been identified, we suggest that there are opportunities to extend the limit of dehydration tolerance in Populus.