Recent studies of plant water uptake assume that soil water isotopic composition can be used to infer soil water mobility. However, the strength of the relationship between mobility and isotopic composition remains poorly constrained. In addition, many ecohydrologic investigations are restricted by low sampling frequencies and insufficient soil moisture and matric potential data to support assumptions of soil water mobility. We sampled bulk soil water every 14 to 21 days in hillslope and riparian profiles during the 2016 and 2017 growing seasons in a semi-arid watershed outside Boise, ID. We collected twig samples of four tree and shrub species concurrently. Plant and soil water samples extracted by cryogenic vacuum distillation were analyzed for δ2H and δ18O composition. We installed volumetric water content and soil matric potential sensors at five and four depths in the hillslope profile, respectively. Shallow bulk soil water became progressively enriched in both isotopes as mobility declined over the two growing seasons, particularly at the hillslope site. Strong correlations existed between isotopic composition and mobility in shallow layers but isotopic composition alone failed to predict soil water mobility. No relationship existed in deeper soil water, suggesting water loss only through transpiration and drainage. We propose that evaporation depth is a strong control on the relationship between soil water mobility and isotopic composition. Plant water isotope evolution suggests that Douglas Fir relies on deeper water sources than sagebrush or chokecherry. These results underscore the utility of measurement of soil water mobility proxies in future ecohydrologic studies