Photosystem I (PSI) is one of two the photosystems involved in oxygenic photosynthesis. PSI of cyanobacteria exists in monomeric, trimeric, and tetrameric forms, which is in contrast to the strictly monomeric form of PSI in plants and algae. The tetrameric organization raises questions about its structural, physiological, and evolutional significance. Here we report the ~3.9 Å resolution cryo-EM structure of tetrameric PSI from the thermophilic, unicellular cyanobacterium Chroococcidiopsis sp. TS-821. The structure resolves all 44 subunits and 448 cofactor molecules. We conclude that the tetramer is arranged via two different interfaces resulting from a dimer-of-dimers organization. The localization of chlorophyll molecules permits an excitation energy pathway within and between adjacent monomers. Bioinformatics analysis reveals conserved regions in PsaL subunit that correlate with the oligomeric state. Tetrameric PSI may function as a key evolutionary step between the trimeric and monomeric forms of PSI organization in photosynthetic organisms.