Diazotrophic microorganisms alleviate nitrogen limitation at marine cold seeps using nitrogenase, encoded in part by the genenifH. Here, we investigatednifH-containing organisms (NCOs) inside and outside six biogeochemically heterogeneous seeps using amplicon sequencing and quantitative real-time PCR (qPCR) ofnifHgenes and transcripts. We detectednifHgenes affiliated with anaerobic methane-oxidizing ANME-2 archaea and sulfate-reducing Desulfobacteraceae, consistent with previous studies, but also phylogenetically and likely metabolically diverse organisms, includingDesulfoglaeba,CandidatusMethanoliparia, and Desulfuromonadales. In total, we recovered 10,734 bona fidenifHsequence variants affiliated with 18 bacterial and archaeal phyla (17 within seeps), a subset of which were transcribed at nearly all seeps investigated. We corrected our qPCR data based on our amplicon results, which found that 71% of recovered sequences were not bona fidenifH,and we recommend a similar correction in future qPCR studies that use broadnifHprimers.NifHabundance was up to three orders of magnitude higher within seeps, was highly correlated withmcrAgene abundance, and, when corrected, was negatively correlated with porewater ammonium