Thylakoid membranes scaffold an assortment of large protein complexes that work together to harness the energy of light to produce oxygen, NADPH, and ATP. It has been a longstanding challenge to visualize how the intricate thylakoid network organizes these protein complexes to finely tune the photosynthetic reactions. Using cryo-electron tomography to analyze membrane surface topology, we have mapped the native molecular landscape of thylakoid membranes within green algae cells. Our tomograms provide insights into the molecular forces that drive thylakoid stacking and reveal that photosystems I and II are strictly segregated at the borders between appressed and non-appressed membrane domains. This new approach to charting thylakoid topology lays the foundation for dissecting photosynthetic regulation at the level of single protein complexes within the cell.