Background/Question/Methods Variation in stress tolerance is well documented across not only species, but also across populations. The greatest intraspecific variation tends to be observed in species with large or expanding ranges. In plants, variability in traits like drought tolerance may be one mechanism by which populations become established in new areas. Here we used two closely related conifer species (Juniperus virginiana and J. scopulorum) to investigate trait variation in J. virginiana across its broad and expanding range. We compared water use traits from populations of J. virginiana sourced across a thermal and precipitation gradient to those of J. scopulorum, which is found primarily in arid sites. We predicted that J. virginiana populations from more arid sites would exhibit traits more like J. scopulorum than J. virginiana from humid sites. We quantified the drought tolerance of three populations of J. virginiana and one population of J. scopulorum by measuring mid-day leaf water potential (Ψl) and stomatal conductance (gs) as they were exposed to a soil dry down experiment. We then constructed a vulnerability curve for gs using the “plantecophys” package in R and calculated the point of stomatal closure (Pgs88), populations were considered different if bootstrapped confidence intervals did not overlap. Results/Conclusions Juniperus virginiana from the most arid location showed the greatest response to water availability in the dry down experiment, having the greatest gs at high water potentials but declining rapidly as soils dried. The plants showed a linear decline in Ψl and reached a point where and gs was near zero between 17 and 21 days. The mean minimum Ψl at the end of the experiment was dependent on the source site (ANOVA F3,12 = 12.31, p < 0.001), and a posthoc analysis showed that Ψl decreased with increasing site aridity with a minimum Ψl of -5.45 ± 0.16 (SE) MPa for J. scopulorum to -2.97 ± 0.25 (SE) for J. virginiana from the most humid site. The populations also exhibited different Pgs88, ranging from -3.4 MPa in J. scopulorum to -1.6 MPa for J. virginiana from the most humid site. These results show that there was a correlation between leaf level water use traits and source climate for J. virginiana, with populations from more arid sites rapidly declining in gs but maintaining some level of gs at lower Ψl. This variation highlights the importance of understanding intraspecific variation when predicting responses of woody plants to climactic stress factors.