Abstract Biological nitrogen fixation plays a critical role in marine primary production, yet, our understanding of marine N 2 -fixers (diazotrophs) is hindered by limited observations. Here, we developed a quantitative image analysis pipeline in concert with mapping of molecular markers for mining >2,000,000 images and >1,300 metagenomes in surface, deep chlorophyll maximum and mesopelagic samples across 6 size fractions (<0.2-2000 μm). Imaging and PCR-free molecular data were remarkably congruent. Sequences from diazotrophs were detected from the ultrasmall bacterioplankton (<0.2 μm) to mesoplankton (180-2000 μm), while images predicted symbiotic and colonial-forming diazotrophs (>20 µm). Imaging and molecular data estimated that polyploidy can significantly impact gene abundances of symbiotic vs colonial-forming diazotrophs. In general our results support the canonical view that larger sized diazotrophs (>10 μm) dominate the tropical belts, while sequences from unicellular cyanobacterial and non-cyanobacterial diazotrophs were globally distributed in surface and the mesopelagic. Co-occurring diazotrophic lineages of different lifestyles were frequently encountered, and several new high density regions of diazotrophs were identified in the global ocean. Overall, this work provides an update of marine diazotroph biogeographical diversity and contributes a new bio-imaging-informatic workflow.