Photosynthetic efficiency is a critical determinant of crop yield potential, though it remains below the theoretical optimum in modern crop varieties. Enhancing mesophyll conductance, i.e. the rate of carbon dioxide diffusion from substomatal cavities to the sites of carboxylation, may increase photosynthetic and water use efficiencies. To improve water-use-efficiency mesophyll conductance should be increased without concomitantly increasing stomatal conductance. Here we partition variance in mesophyll conductance to within and among cultivar components across soybeans grown under both controlled and field conditions, and examine the covariation of mesophyll conductance with photosynthetic rate, stomatal conductance, water-use-efficiency and leaf mass per area. We demonstrate that mesophyll conductance varies more than 2-fold and that 38% of this variation is due to cultivar identity. As expected mesophyll conductance is positively correlated with photosynthetic rates. However, a strong positive correlation between mesophyll and stomatal conductance among cultivars apparently impedes positive scaling between mesophyll conductance and water-use-efficiency in soybean. Contrary to expectations, photosynthetic rates and mesophyll conductance both increased with increasing leaf mass per area. The presence of genetic variation for mesophyll conductance suggests there is potential to increase photosynthesis and mesophyll conductance by selecting for greater leaf mass per area. Increasing water-use-efficiency though, is unlikely unless there is simultaneous stabilizing selection on stomatal conductance.