Summary The oxygen stable isotope composition of plant organic matter (OM) (particularly of wood and cellulose in the tree ring archive) is valuable in studies of plant–climate interaction, but there is a lack of information on the transfer of the isotope signal from the leaf to heterotrophic tissues. We studied the oxygen isotopic composition and its enrichment above source water of leaf water over diel courses in five tree species covering a broad range of life forms. We tracked the transfer of the isotopic signal to leaf water‐soluble OM and further to phloem‐transported OM. Observed leaf water evaporative enrichment was consistent with values predicted from mechanistic models taking into account nonsteady‐state conditions. While leaf water‐soluble OM showed the expected 18O enrichment in all species, phloem sugars were less enriched than expected from leaf water enrichment in Scots pine (Pinus sylvestris), European larch (Larix decidua) and Alpine ash (Eucalyptus delegatensis). Oxygen atom exchange with nonenriched water during phloem loading and transport, as well as a significant contribution of assimilates from bark photosynthesis, can explain these phloem 18O enrichment patterns. Our results indicate species‐specific uncoupling between the leaf water and the OM oxygen isotope signal, which is important for the interpretation of tree ring data.