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Abstract Successful use of stable isotopes (δ2H and δ18O) in ecohydrological studies relies on the accurate extraction of unfractionated water from different types of soil samples. Cryogenic vacuum distillation (CVD) is a common laboratory‐based technique used for soil water extraction; however, the reliability of this technique in reflecting soil water δ2H and δ18O is still of concern. This study examines the reliability of a newly developed automatic cryogenic vacuum distillation (ACVD) system. We further assessed the impacts of extraction parameters (i.e. extraction time, temperature and vacuum) and soil properties on the recovery of soil water δ2H and δ18O for the ACVD and traditional cryogenic vacuum distillation (TCVD) systems. Finally, we investigated the potential influence of CVD (ACVD and TCVD) technique on the prediction of plant water uptake through a sensitivity analysis. Both ACVD and TCVD similarly extracted water from the rewetted soils, but none of the CVD systems successfully recovered the isotopic signatures of doped water from soil materials. Mean δ2H offsets of extracted soil water were −2.6 ± 1.3‰ and −2.4 ± 1.7‰ for ACVD and TCVD, respectively, while mean δ18O offsets were −0.16 ± 0.14‰ and −0.39 ± 0.37‰. The isotopic offsets of CVD systems were positively correlated with soil clay content, and negatively correlated with soil water content. Using corrected soil data (with CVD offsets) could improve the prediction of plant water uptake based on its high correlation with the environmental factors. This study identifies the isotopic offsets of CVD systems (i.e. ACVD and TCVD) and provides possible solutions for better predicting plant water sources. Even though, the wide use of CVD techniques probably induce noticeable uncertainties in the prediction of plants water uptake depths. The dataset of soil water extraction in this study will have implications for the technological development of CVD techniques.