Abstract The carbon isotope composition of respired CO2 (δ13CR) and bulk organic matter (δ13CB) of various plant compartments informs about the isotopic fractionation and substrate of respiratory processes, which are crucial to advance the understanding of carbon allocation in plants. Nevertheless, the variation across organs, species and seasons remains poorly understood. Cavity Ring-Down Laser Spectroscopy was applied to measure δ13CR in leafy shoots and woody stems of maple (Acer platanoides L.), oak (Quercus robur L.) and cedar (Thuja occidentalis L.) trees during spring and late summer. Photosynthesis, respiration, growth and non-structural carbohydrates were measured in parallel to evaluate potential drivers for respiratory fractionation. The CO2 respired by maple and oak shoots was 13C-enriched relative to δ13CB during spring, but not late summer or in the stem. In cedar, δ13CR did not vary significantly throughout organs and seasons, with respired CO2 being 13C-depleted relative to δ13CB. Shoot δ13CR was positively related to leaf starch concentration in maple, while stem δ13CR was inversely related to stem growth. These relations were not significant for oak or cedar. The variability in δ13CR suggests (i) different contributions of respiratory pathways between organs and (ii) seasonality in the respiratory substrate and constitutive compounds for wood formation in deciduous species, less apparent in evergreen cedar, whose respiratory metabolism might be less variable.