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Summary Nitrogen fixation, the biological conversion of N₂to NH₃, is critical to alleviating nitrogen limitation in many marine ecosystems. To date, few measurements exist of N₂fixation in deep‐sea sediments. Here, we conducted > 400 bottle incubations with sediments from methane seeps, whale falls and background sites off the western coast of the United States from 600 to 2893 m water depth to investigate the potential rates, spatial distribution and biological mediators of benthic N₂fixation. We found that N₂fixation was widespread, yet heterogeneously distributed with sediment depth at all sites. In some locations, rates exceeded previous measurements by > 10×, and provided up to 30% of the community anabolic growth requirement for nitrogen. Diazotrophic activity appeared to be inhibited by pore water ammonium: N₂fixation was only observed if incubation ammonium concentrations were ≤ 25 μM, and experimental additions of ammonium reduced diazotrophy. In seep sediments, N₂fixation was dependent on CH₄and coincident with sulphate reduction, consistent with previous work showing diazotrophy by microorganisms mediating sulphate‐coupled methane oxidation. However, the pattern of diazotrophy was different in whale‐fall and associated reference sediments, where it was largely unaffected by CH₄, suggesting catabolically different diazotrophs at these sites.