Accurate estimates of the δ13C value of CO2 respired from roots (δ13CR_root) and leaves (δ13CR_leaf) are important for tracing and understanding changes in C fluxes at the ecosystem scale. Yet the mechanisms underlying temporal variation in these isotopic signals are not fully resolved. We measured δ13CR_leaf, δ13CR_root, and the δ13C values and concentrations of glucose and sucrose in leaves and roots in the C4 grass Sporobolus wrightii and the C3 tree Prosopis velutina in a savanna ecosystem in southeastern Arizona, USA. Night-time variation in δ13CR_leaf of up to 4.6 ± 0.6‰ in S. wrightii and 3.0 ± 0.6‰ in P. velutina were correlated with shifts in leaf sucrose concentration, but not with changes in δ13C values of these respiratory substrates. Strong positive correlations between δ13CR_root and root glucose δ13C values in P. velutina suggest large diel changes in δ13CR_root (were up to 3.9‰) influenced by short-term changes in δ13C of leaf-derived phloem C. No diel variation in δ13CR_root was observed in S. wrightii. Our findings show that short-term changes in δ13CR_leaf and δ13CR_root were both related to substrate isotope composition and concentration. Changes in substrate limitation or demand for biosynthesis may largely control short-term variation in the δ13C of respired CO2 in these species.