In a two-year field trial, the effect of nitrogen (N) and sulfur (S) fertilization was investigated on grain yield, grain quality parameters, formation of acrylamide (AA), and the precursor free asparagine (Asn) in organically and conventionally produced winter wheat cultivars. In both production systems, different types, amounts, and temporal distributions of N were tested. While the effect of S fertilizer types and amounts on free Asn was only tested in the conventional farming system. Within both cropping systems, grain yield and baking quality were significantly influenced by N treatment while the effect on free Asn was only minor. Especially within the organic farming system, increasing N fertilization levels did not increase free Asn significantly. A slight trend of increasing free Asn levels with an intensified N supply was observed, especially in the presence of crude protein contents of 14% or higher. But only N amounts of 180 kg N ha⁻¹ or higher increased the probability of high free Asn contents considerably, while N supply below that amount led to free Asn values similar to the unfertilized controls. The results indicated that good baking quality can be achieved without significantly increasing free Asn levels. In addition, cultivars affected the levels of free Asn significantly. Compared to cv. “Bussard” and “Naturastar”, cv. “Capo” exhibited the lowest AA formation potential at an N supply of 180 kg N ha⁻¹ while simultaneously reaching a crude protein content > 15% (conventional) and > 12% (organic). Thus, it seems that cultivars differ in their ability to store and incorporate free Asn into proteins. Over all trials, a correlation of free Asn and AA was shown by R² = 0.77, while a relation of free Asn and protein was only R² = 0.36. Thus, lowering free Asn by adjusting N treatments should not necessarily affect baking quality. S nutrition within conventional farming did not change free Asn amount or crude protein significantly, probably due to the fact that soil was not sulfate-deficient. In summary, it was evident that free Asn amounts in wheat varied widely both within cultivars and between cropping systems. In order to clearly unravel genotypic differences and their interaction with environmental factors and especially N fertilization, further research is needed.