Cultivar, habitat conditions and agrotechnology have an influence on the yield and chemical composition of rye grain. The main anti-nutritional substances present in rye grain include alkylresorcinols, water-soluble pentosans and trypsin inhibitors. The aim of this study was to determine the variability in yield and the concentration of anti-nutritional compounds in the grain of selected winter rye cultivars in relation to nitrogen fertilisation levels and weather conditions. Field studies were conducted at the Experimental Station of IUNG-PIB in Osiny (Poland) in two growing seasons (2018/2019 and 2019/2020). The experiment was located on pseudo-polylic soil using the randomised sub-block method in three replications. The first factor of the experiment was the level of nitrogen fertilisation (0, 70 and 140 kg N∙ha−1) and the second was the population (Dańkowskie Skand, Piastowskie) and hybrid (KWS Vinetto, SU Performer) winter rye cultivars. The study showed that the yield of winter rye depended on the genotype and the level of nitrogen fertilisation. The hybrid cultivars yield ed 17.9% higher in relation to the population cultivars. The content of anti-nutritional compounds in rye grain depended significantly on genotype, level of nitrogen fertilisation and weather conditions. The reason for the higher synthesis of anti-nutrients in rye grain was the stressful weather conditions occurring in the 2019/2020 season. Nitrogen fertilisation influenced the content of alkylresorcinols, water-soluble pentosans and trypsin inhibitor activity in grain. The interaction of cultivar and fertilisation was also found to shape the content of the aforementioned anti-nutrients.