The growing demand for global food production is likely to be a defining issue facing humanity over the next 50 years. In order to tackle this challenge, there is a desire to bioengineer crops with higher photosynthetic efficiencies, to increase yields. Recently, there has been a growing interest in engineering leaves with higher mesophyll conductance (g_m), which would allow CO₂ to move more efficiently from the substomatal cavities to the chloroplast stroma. However, if crop yield gains are to be realised through this approach, it is essential that the methodological limitations associated with estimating gm are fully appreciated. In this review, we summarise these limitations, and outline the uncertainties and assumptions that can affect the final estimation of g_m. Furthermore, we critically assess the predicted quantitative effect that elevating g_m will have on assimilation rates in crop species. We highlight the need for more theoretical modelling to determine whether altering g_m is truly a viable route to improve crop performance. Finally, we offer suggestions to guide future research on g_m, which will help mitigate the uncertainty inherently associated with estimating this parameter.