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ABSTRACT Photoautotrophic diazotrophs, specifically the generaTrichodesmiumand UCYN-A, play a pivotal role in marine nitrogen cycling through their capacity for nitrogen fixation. Despite their global distribution, the microdiversity and environmental drivers of these diazotrophs remain underexplored. This study provides a comprehensive analysis of the global diversity and distribution ofTrichodesmiumand UCYN-A using the nitrogenase gene (nifH) as a genetic marker. We sequenced 954 samples from the Pacific, Atlantic, and Indian Oceans as part of the Bio-GO-SHIP project. Our results reveal significant phylogenetic and biogeographic differences between and within the two genera.Trichodesmiumexhibited greater microdiversity compared to UCYN-A, with clades showing region-specific distribution.Trichodesmiumclades were primarily influenced by temperature and nutrient availability. They were particularly frequent in regions of phosphorus stress. In contrast, UCYN-A was most frequently observed in regions experiencing iron stress. UCYN-A clades demonstrated more homogeneous distributions, with a single sequence variant within the UCYN-A1 clade dominating across varied environments. The biogeographic patterns and environmental correlations ofTrichodesmiumand UCYN-A highlight the role of microdiversity in their ecological adaptation and reflect their different ecological strategies. These findings underscore the importance of characterizing the global patterns of fine-scale genetic diversity to better understand the functional roles and distribution of marine nitrogen-fixing photoautotrophs.IMPORTANCEThis study provides insights into the global diversity and distribution of nitrogen-fixing photoautotrophs, specificallyTrichodesmiumand UCYN-A. We sequenced 954 oceanic samples of thenifHnitrogenase gene and uncovered significant differences in microdiversity and environmental associations between these genera.Trichodesmiumshowed high levels of sequence diversity and region-specific clades influenced by temperature and nutrient availability. In contrast, UCYN-A exhibited a more uniform distribution, thriving in iron-stressed regions. Quantifying these fine-scale genetic variations enhances our knowledge of their ecological roles and adaptations, emphasizing the need to characterize the genetic diversity of marine nitrogen-fixing prokaryotes.