Plasmodesmata (PD) are nanochannels that facilitate cell-to-cell transport in plants. PD can form either as a result of endoplasmic reticulum being trapped between developing daughter cells during cell division (primary PD) or de novo through recruitment of Golgi bodies into the cell wall (secondary PD). In a leaf, which is the main photosynthetic organ in plants, PD are crucial for metabolite and sugar transport. More productive and photosynthetically efficient C4 monocot crops, like corn and sorghum, form more PD in their leaves than their less efficient C3 relatives like rice and wheat. In C4 leaves, PD play an essential role in facilitating the rapid metabolite exchange between the mesophyll (M) and bundle sheath (BS) cells to operate a biochemical CO2 concentrating mechanism, which increases the CO2 partial pressure at the site of Rubisco in the BS cells and hence photosynthetic efficiency. The genetic mechanism controlling PD formation in C3 and C4 leaves is largely unknown, especially in monocot crops, due to the technical challenge of quantifying these nanostructures with electron microscopy. To address this issue, we have generated stably transformed lines of Oryza sativa (rice, C3) and Setaria viridis (setaria, C4) with fluorescent protein-tagged PD to build the first spatiotemporal atlas of leaf PD density in monocots without the need for laborious transmission electron microscopy. We performed complementary temporal mRNA sequencing and gene co-expression network analysis to identify potential PD formation genes in C3 and C4 monocot leaves. Select gene modules and PD-associated genes identified from our gene co-expression network analysis can be used for functional validation in C3 and C4 crops. This will provide an opportunity to manipulate cell-to-cell transport and improve photosynthetic efficiency in leaves of economically valuable grasses.