Thylakoid membranes carry out photosynthetic electron transport and represent the most complex energy-transducing membranes. Despite a profound knowledge on their structure and function, very little is known on how this supramolecular machinery is put together during the biogenesis of thylakoids. The biogenesis process includes the highly-ordered, step-wise assembly of proteins, lipids, pigments and inorganic co-factors which to a large extent is mediated by dedicated assembly factors assisting specific steps in the biogenesis pathway. Recent work suggests that thylakoid biogenesis is additionally subcompartmentalized at the spatial level in that it initiates at distinct membranous substructures and proceeds to generate functional, highly ordered membrane systems in a still largely unknown manner. Here, a multidisciplinary consortium that combines unique expertise in molecular genetics, biochemistry, biophysics and ultrastructural analyses proposes to take a systematic approach towards elucidating the molecular details and the spatiotemporal sequence of events leading to the formation of functional thylakoids. As during evolution, the complexity of thylakoid organization has increased dramatically, a set of suitable model organisms will be comparatively studied to follow the complete evolutionary path from simple photosynthetic membrane patches in primitive cyanobacteria to elaborate thylakoid networks in chloroplasts of vascular plants.