The temperature responses of mesophyll conductance (gₘ) were investigated for nine species using carbon isotope techniques combining tunable diode laser spectroscopy and gas exchange measurements. Species included the evergreen trees Eucalyptus pauciflora and Quercus engelmannii; the tropical evergreen tree Lophostemon confertus; as well as the herbaceous species Nicotiana tabacum, Oryza sativa, Triticum aestivum, Gossypium hirsutum, Glycine max and Arabidopsis thaliana. Responses varied from a two‐ to threefold increase in mesophyll conductance between 15 and 40 °C observed for N. tabacum, G. hirsutum, G. max and E. pauciflora to almost no change in L. confertus and T. aestivum. To account for the different temperature responses between species, we suggest that there must be variation in both the activation energy for membrane permeability and the effective pathlength for liquid phase diffusion. Stomatal conductance was relatively independent of increases in leaf temperature and concomitant increases in leaf to air vapour pressure difference. Two exceptions were Eucalyptus and Gossypium, where stomatal conductance increased with temperature up to 35 °C despite increasing leaf to air vapour pressure. For a given species, temperature responses of stomatal and mesophyll conductance were independent of one another.