• The first broad species survey of diurnal variation in carbon (C) isotope signatures of leaf dark-respired CO2 (δ13Cres) is presented here and functional differences and diurnal dynamics are linked to fractionation in different respiratory pathways, based on 13C-labelling experiments. • δ13Cres was analysed with a rapid in-tube incubation technique in 16 species. • A large diurnal increase in δ13Cres (4–8‰) occurred in evergreen, slow-growing and aromatic species and correlated significantly with cumulative photosynthesis, whereas no variation occurred in herbaceous, fast-growing plants or temperate trees. The diurnal increase in δ13Cres declined almost proportionally to reductions in cumulative light and was reduced in growing compared with mature leaves. • Pyruvate positional labelling provided direct evidence that functional groups differ in C allocation between respiratory pathways owing to different metabolic demands for growth, maintenance and secondary metabolism. Diurnal increase in C flux through pyruvate dehydrogenase (for investment in, for example, isoprene or aromatic compounds) combined with consistently low Krebs cycle activity resulted in pronounced increase in δ13Cres in evergreen and aromatic species. By contrast, fast growing herbs with high respiratory demand exhibited no diurnal changes since C was fully respired. Hence, diurnal δ13Cres pattern may provide information for C allocation in plants