Soybean ( Glycine max [L.] Merr) is among the most important agricultural seed crops and source of vegetable protein. Further yield improvements per unit land area are needed to meet future demand and avoid destruction of more natural lands. Mesophyll conductance ( g_m) in C ₃ crops quantifies the ease with which CO ₂ can transfer from the sub-stomatal cavity to Rubisco within the mesophyll. Increasing g_m is in theory most attractive as it would increase photosynthesis, yield potential and water-use efficiency. Most measurements of g_m have been made during steady-state light saturated photosynthesis. However, in field crop canopies, light fluctuations are frequent and the speed with which g_m can increase following shade to sun transitions is likely important to crop carbon gain. Is there variability in g_m that could be used in breeding? If so, indirect selection could be expected to have already increased g_m and be apparent when comparing wild ancestors of soybean ( Glycine soja) to a domesticated high-yielding cultivar. The elite LD11 was compared with four ancestor accessions collected from the assumed area of domestication by concurrent measurements of gas exchange and carbon isotope discrimination (∆ ¹³C). This allowed estimation of g_m both through induction following transfer to high light and at steady-state. The results have shown 1) gm was a significant limitation to soybean photosynthesis both at steady-state and through light induction, especially when the major biochemical limitation was in vivo Rubisco activity and, 2) compared to the ancestral accessions, the elite LD11 showed a large and significant increase in g_m at both steady-state and through light induction, which also corresponded to a substantial increase in leaf level CO2 assimilation and water use efficiency.