A warming climate combined with frequent and severe fires cause permafrost to thaw, especially in the region of discontinuous permafrost, where soil temperatures may only be a few degrees below 0 °C. Soil thaw releases carbon and nitrogen into the actively cycling pools, and whereas C emissions following permafrost thaw are well documented, the fates of N remain unclear. Denitrification could release N from ecosystems as nitrous oxide or nitrogen gas, but the contributions of these processes to the high-latitude N cycle remain uncertain. We quantified microbial capacity for denitrification and N2O production in boreal soils, lakes, and streams using anoxic C- and N-amended assays, and assessed correlates of denitrifying enzyme activity in Interior Alaska. Riparian soils and stream sediments supported the highest potential rates of denitrification, upland soils were intermediate, and lakes supported lower rates, whereas deep permafrost soils supported little denitrification. Time since fire had no effect on denitrification potential in upland soils. Across all landscape positions, DEA was negatively correlated with ammonium pools. Within each landscape position, potential rate of denitrification increased with soil or sediment organic matter content. Widespread N loss to denitrification in boreal forests could constrain the capacity for N-limited primary producers to maintain C stocks in soils following permafrost thaw.