Isoprene emission potential (ES) varies in tree canopies, and such variations have potentially major implications for predicting canopy level emissions. So far, quantitative relationships of ES with irradiance are missing, and interspecific variation in ES plasticity and potential effects on canopy level emissions have not been characterized. ES, foliage structural, chemical, and photosynthetic characteristics were studied relative to integrated within-canopy daily quantum flux density (Qint) in temperate deciduous tree species Quercus robur, Populus tremula, Salix alba, and Salix caprea, and canopy isoprene emissions were calculated considering observed variation in ES and under different simplifying assumptions. Strong positive curvilinear relationships between nitrogen and dry mass per unit area, photosynthetic potentials and ES per area with Qint were observed. Structural, chemical, and photosynthetic traits varied 1.5-fold to 4-fold and ES per area 3-fold to 27-fold within the canopy. ES variation reflected accumulation of mesophyll cell layers and greater emission capacity of average cells. Species with largest structural and photosynthetic plasticity had greatest plasticity in ES. Relative to the simulation considering within-canopy variation in ES, the bias from assuming a constant ES varied between −8% and +68%, and it scaled positively with ES plasticity. The bias of big-leaf simulations varied between −22% and −35%, and it scaled negatively with ES plasticity. A generalized canopy response function of ES developed for all species resulted in the lowest bias between −11% and 6% and can be recommended for practical applications. The results highlight huge within-canopy and interspecific variation in ES and demonstrate that ignoring these variations strongly biases canopy emission predictions.