Mesophyll conductance (g ) is the diffusion of CO from intercellular air spaces (IAS) to the first site of carboxylation in the mesophyll cells. In C species, g is influenced by diverse leaf structural and anatomical traits; however, little is known about traits affecting g in C species. To address this knowledge gap, we used online oxygen isotope discrimination measurements to estimate g and microscopy techniques to measure leaf structural and anatomical traits potentially related to g in 18 C grasses. In this study, g scaled positively with photosynthesis and intrinsic water-use efficiency (TE ), but not with stomatal conductance. Also, g was not determined by a single trait but was positively correlated with adaxial stomatal densities (SD ), stomatal ratio (SR), mesophyll surface area exposed to IAS (S ) and leaf thickness. However, g was not related to abaxial stomatal densities (SD ) and mesophyll cell wall thickness (T ). Our study suggests that greater SD and SR increased g by increasing S and creating additional parallel pathways for CO diffusion inside mesophyll cells. Thus, SD , SR and S are important determinants of C -g and could be the target traits selected or modified for achieving greater g and TE in C species.