The challenges of the global food supply and environment conservation require ongoing scientific observations of soil-to-plant and plant-to-environment interactions with the aim of improving agriculture resource management. This study included observations of winter wheat yield and biomass of four varieties over three consecutive growing seasons and four site-year cases to assess the effects of nitrogen (N) fertilization rate and time of application on grain yield and biomass. For different wheat varieties, the full factorial design was performed, where factorial combinations of year, location, fall and spring N applications were laid out in a randomized complete block design. The N rate significantly influenced grain yield and biomass production efficiency. The time of N application had a highly significant effect on grain yield, biomass and NUE traits. The N rate of 120 kg ha−1 was recognized as a breakpoint over which the grain yield and biomass showed a downtrend. N application in the fall had a significantly higher impact on grain yield and biomass compared to spring N application. The major contribution of wheat variability production belongs to seasonal climate circumstances (<85%) and consequential intrinsic soil properties. The average difference of grain yield between varieties was 15.75%, and 12% of biomass, respectively.