Our study investigated the seasonal variability in historical water source use of Fraxinus excelsior and Populus nigra. We utilized a new method which combines tree-ring isotopes and isotope modelling to determine how tree water use varied at the sub-annual level over 10 consecutive growing seasons (2000-2010). We conducted tree-ring micro-slicing to produce highly resolved cellulose δ18O records and back calculated the δ18O signature of the source water used for growth of each micro-slice. We compared these results to regional water δ18O signatures to infer seasonal hydrological partitioning. Fraxinus was dependent on non-growing and growing season rainfall from the vadose zone whilst Populus relied on hyporheic and regional groundwater. We observed a convergence in water use of both species from 2007 which is attributed to a decline in phreatic water access to Populus