<p>Cyanobacteria are the only prokaryotic group capable of performing oxygenic photosynthesis and nitrogen fixation (catalyzed by extremely oxygen-sensitive nitrogenase) simultaneously. A systematic understanding of the phylogenetic distribution of nitrogen-fixing ability remains incomplete due to difficulties in accurately identifying genes essential for nitrogen fixation from genomic data, caused by occasional confusion with nitrogenase-like enzymes and fragmentation of <i>nif</i> genes in heterocystous species. In this study, we comprehensively evaluated the distribution of the minimal nitrogen fixation gene set (<i>nifHDKENB</i>) across 586 major cyanobacterial strains. Overall, 48% of the strains were predicted to be nitrogen-fixing with a mosaic distribution in non-heterocystous strains. Using the results as reference data, we evaluated nitrogen fixation across a broader range of cyanobacterial lineages, including uncultured strains and metagenomes. We predicted that approximately 20% of 2,718 strains are nitrogen fixers, revealing a high <i>nif</i> retention rate of <i>nif</i> genes in the Cyanobacteria comparing with other bacterial phyla. While most cyanobacteria carry Group I <i>nif</i> genes, we identified widely distributed lineages with Group II <i>nif</i> genes, some of which are associated with distinct CnfR proteins. We discuss the dynamic evolutionary history of <i>nif</i> genes in cyanobacteria.</p>