Significance Cyanobacteria are model organisms for photosynthesis in the laboratory, are key producers of the chemical energy that drives life, and are being developed as biofuel and chemical producers for industry. Despite the importance of these organisms for environmental and biotechnological applications, only a small percentage of cyanobacterial genes and intergenic regions have been experimentally evaluated for their impact on the organisms’ survival. Here, we present experimental analysis of the complete set of genomic regions necessary for survival in a cyanobacterium achieved by screening for the fitness of hundreds of thousands of mutants. In addition to improving our fundamental understanding of Cyanobacteria, this research more broadly provides a snapshot of the essential genes and intergenic regions necessary to live the photosynthetic lifestyle.