Global gaseous nitrogen (N2) fixation rates may be underestimated and data is lacking from many regions without conspicuous diazotrophic cyanobacteria, such as cold oceans. We estimated N2 fixation rates at diverse sites in the Canadian Arctic, including the mouth of the Mackenzie River, the offshore Beaufort Sea, Lancaster Sound, Baffin Bay and a river influenced fjord. We also identified potential diazotrophic communities using a targeted survey of the nifH gene. Nitrogen fixation rates ranged from 0.02 nmol N L−1 d−1 in Baffin Bay to 4.45 nmol N L−1 d−1 in the Mackenzie River plume. Sequences recovered from the nifH gene survey belonged mainly to Cluster III, a group of nifH sequences associated with diverse microorganisms, with some α- andγ-proteobacterianifH genes at most sites. Cyanobacteria nifH genes with best matches to Nostocales, which are common in Arctic freshwaters, were recovered from the marine Beaufort Sea. The geographic pattern of N2 fixation rates and nifHgene identities suggest that the Mackenzie River is the source of a diazotrophic community that contributes new nitrogen to the nitrogen-depleted surface waters of the Beaufort Sea. This first record of N2 fixation at high latitudes refines our understanding of the global nitrogen budget.