Limited information exists on how tillage and nitrogen (N) fertilization affects small-scale variation in nitrogen use efficiency (NUE) and crop performance. In a two-year field study under temperate conditions, we investigated how tillage (NT, no-tillage; CT, conventional tillage) and N fertilization affected the small-scale variation in NUE and winter wheat performance (grain yield, Gw; grain protein concentration, GPC). A randomized complete block design with three replications was used. Within each tillage plot (12 × 35 m2), N rates (0, 50, 100, 150, 200, 250 kg N ha−1) were completely randomized within each of four groups of microplots (1.5 × 1.5 m2). Early-season soil mineral N (Nmin) was also monitored in both years. At rates < 150 kg N ha−1, NT was not competitive with CT in terms of Gw and NUE. Gw and aboveground plant N were not correlated with Nmin prior to application of N fertilizer. NT usually led to larger spatial heterogeneity of Nmin, Gw, and NUE. The small-scale variability of Gw, GPC, NUE, and N supply decreased with increasing N fertilization rates under both tillage systems. Significant increases in Gw and GPC were observed with increasing N rates, whereas NUE decreased slightly with increasing N rates in both NT and CT. The overall moderate spatial variation in Nmin, Gw, and NUE did not justify site-specific N fertilization in these small fields, with the exception of the stony within-plot positions, which were not responsive to rates of N > 50 kg N ha−1.