In cold agricultural regions, animal manure and synthetic fertilizers may be applied in the fall for convenience. However, the fate of applied nitrogen (N) is unclear and may differ depending on N source and interannual and regional variations in winter conditions. A multi-region study using ¹⁵N-labelled reactive N (NH₄-¹⁵N) applied in the fall with pig slurry, dairy cattle slurry, and ammonium sulfate was carried out under a range of climatic conditions. Nitrification and immobilization of applied NH₄-N occurred throughout the winter period at all sites. Transformation and losses were slower and less at the sites where significant soil freezing occurred than at the site where soil rarely froze, highlighting the repressive effect of frost. Nevertheless, losses were similar among sites with significant freezing despite marked differences in duration and extent of freezing. This suggests that soil microbes were adapted to prevailing winter conditions at each site and able to use and transform fall-applied N throughout the winter period. Overall, 47%–94% of fall-applied NH₄-N was lost from the top 30 cm of soil before seeding in the next spring. Losses were generally greater with synthetic fertilizer than manures, likely because fresh carbon added with manures stimulated immobilization of NH₄-N. This multi-region assessment indicates that reactive N applied in the fall has high vulnerability to loss in cold and frozen soils, and strategies for improving N retention over the winter are required even in areas where prolonged freezing occurs.