Abstract The cell wall plays a key role in determining mesophyll conductance ( g m) and photosynthetic capacity. While the impact of wall thickness ( T cw) on g m is well understood, the influence of wall composition and structural interactions on T cw and g m remains unclear, and it is unknown whether these factors have been affected during crop domestication. In this study, we examined 25 wild and 13 domesticated Gossypium genotypes to investigate whether variations in T cw, composition and structure affected g m and photosynthesis. X-ray diffraction was used to analyze internal cell wall structure. Cotton domestication reduced T cw by modifying pectin-to-(cellulose+hemicellulose) ratio and increasing cellulose crystallinity. In contrast, this ratio only correlated with g m in wild genotypes, while in domesticated ones, 1/(pectin+cellulose+hemicellulose) ratio better explained g m variation, reflecting internal property differences of cell wall. Cell wall composition and structure regulate g m differently in wild and domesticated genotypes. In wild genotypes, pectin-to-(cellulose+hemicellulose) ratio influences CO 2 diffusion, while in domesticated ones, pectin may behave structurally, making 1/(pectin+cellulose+hemicellulose) a better predictor. We suggest that the exceptionally low T cw values reported in cotton domesticated genotypes indicate they have reached the lower limit, which may impose physical constraints on component proportions and conformation. Keywords: cell wall component, cell wall thickness, cellulose crystallinity, domestication, mesophyll conductance, pectin