In this study we measured δ¹³C in various carbon pools along the basipetal transport pathway in co-occurring Pinus pinaster and Acacia longifolia trees under Mediterranean climate conditions in the field. Overall, species differences in photosynthetic discrimination resulted in more enriched δ¹³C values in the water-conserving overstory P. pinaster relative to the water-spending understory invasive A. longifolia. Post-photosynthetic fractionation effects resulted in differences in δ¹³C of water-soluble organic matter pools along the plant axis with progressive depletion in δ¹³C from the canopy to the trunk (∼6.5‰ depletion in A. longifolia and ∼0.8‰ depletion in P. pinaster). Regardless of these fractionation effects, phloem sap δ¹³C in both terminal branches and the main stem correlated well with environmental parameters driving photosynthesis for both species, indicating that phloem sap δ¹³C has potential as an integrative tracer of changes in canopy carbon discrimination (Δ¹³C). Furthermore, we illustrate that a simple model based on sap flow estimated canopy stomatal conductance (G(S)) and phloem sap δ¹³C measurements has significant potential as a tool for estimating canopy-level carbon assimilation rates.