This study explored the measurement of 238U/235U ratios in black spruce seedlings as a tracer to investigate the origin of U taken up by this biogeochemical sampling media. Inductively coupled plasma quadrupole mass spectrometer (ICP-QMS) and neutron activation (NAA) analyses of the 238U/235U ratios in the total digested (HF/HNO3-aqua regia) portions of substrates (soil and peat) treated with 200 ml aliquots of 100, 250, and 400 mg/l of aqueous solutions of depleted uranium (DU) exhibited a substantial deviation from the accepted natural value of 137.88. Similarly, the measured 238U/235U ratios in stems of seedlings grown in the DU-dosed substrates ranged from 174 to 210 and are linearly correlated with the dose of DU applied. The elevated 238U/235U ratio in the seedlings suggests that U taken up by the plants is derived predominantly from the labile DU components in water rather than the component adsorbed onto mineral phases in the substrate. Consequently, it was concluded that the U contents in vegetation used as a biogeochemical sampling medium are derived from the labile components in groundwater and/or surface water. The precision and accuracy of the measured ratios were compared with the 238U/235U ratios of in-house granite and uraninite reference samples.