Nitrogen availability limits marine productivity across large ocean regions. Diazotrophs can supply new nitrogen to the marine environment via nitrogen (N2) fixation, relieving nitrogen limitation. The distributions of diazotrophs and N2 fixation have been hypothesized to be generally controlled by temperature, phosphorus, and iron availability in the global ocean. However, even in the North Atlantic where most research on diazotrophs and N2 fixation has taken place, environmental controls remain contentious. Here we measure diazotroph composition, abundance, and activity at high resolution using newly developed underway sampling and sensing techniques. We capture a diazotrophic community shift from Trichodesmium to UCYN-A between the oligotrophic, warm (25–29 °C) Sargasso Sea and relatively nutrient-enriched, cold (13–24 °C) subpolar and eastern American coastal waters. Meanwhile, N2 fixation rates measured in this study are among the highest ever recorded globally and show significant increase with phosphorus availability across the transition from the Gulf Stream into subpolar and coastal waters despite colder temperatures and higher nitrate concentrations. Transcriptional patterns in both Trichodesmium and UCYN-A indicate phosphorus stress in the subtropical gyre. Over this iron-replete transect spanning the western North Atlantic, our results suggest that temperature is the major factor controlling the diazotrophic community structure while phosphorous drives N2 fixation rates. Overall, the occurrence of record-high UCYN-A abundance and peak N2 fixation rates in the cold coastal region where nitrate concentrations are highest (~200 nM) challenges current paradigms on what drives the distribution of diazotrophs and N2 fixation.