The photosynthetic efficiency of plants in different environments is controlled by stomata, hydraulics, biochemistry, and mesophyll conductance (gm). Recently, gm was demonstrated to be the key limitation of photosynthesis in gymnosperms. Values of gm across gymnosperms varied over 20-fold, but this variation was poorly explained by robust structure-bound integrated traits such as leaf dry mass per area. Understanding how the component structural traits control gm is central for identifying the determinants of variability in gm across plant functional and phylogenetic groups. Here, we investigated the structural traits responsible for gm in 65 diverse gymnosperms. Although the integrated morphological traits, shape, and anatomical characteristics varied widely across species, the distinguishing features of all gymnosperms were thick mesophyll cell walls and low chloroplast area exposed to intercellular airspace (Sc/S) compared with angiosperms. Sc/S and cell wall thickness were the fundamental traits driving variations in gm across gymnosperm species. Chloroplast thickness was the strongest limitation of gm among liquid-phase components. The variation in leaf dry mass per area was not correlated with the key ultrastructural traits determining gm. Thus, given the absence of correlating integrated easy-to-measure traits, detailed knowledge of underlying component traits controlling gm across plant taxa is necessary to understand the photosynthetic limitations across ecosystems.