Increasing yield and grain protein concentration (GPC) in wheat (Triticum aestivum L.) without excessive nitrogen (N) rates requires increasing N use efficiency (NUE, yield per available N). We assessed the effects of N rate and timing on yield, GPC, and N nutritional indices of two winter wheat genotypes with similar yield but contrasting GPC. Factorial field experiments evaluated the wheat genotypes ‘LCS Chrome’ (high GPC) and ‘WB–Grainfield’ (low GPC), three N rates, and four N timings (‘Fall’, 100% of N rate applied at Zadoks GS20; ‘Spring’, 100% at GS25; ‘Split’, 40% Fall plus 60% Spring; and ‘Anthesis’, 40% fall, 50% spring, and 10% at GS61) in six Kansas environments. Yield ranged from 2,853 to 8,023 kg ha‐1 and GPC from 88 to 152 g kg‐1. Fall and spring N timing had the lowest and greatest yield difference from the zero‐N control and showed linear, linear‐plateau or no responses to N rate. ‘LCS Chrome’ had 5 to 19 g kg‐1 greater GPC than ‘WB–Grainfield’, which was associated with greater post‐anthesis N uptake (24 vs 15% of maturity‐N uptake), a greater spike N gain between anthesis and maturity (7.4 vs 6.5 g m‐2), and greater N recovery efficiency (0.34 vs 0.28 kg kg‐1). Greater N rates and Anthesis or Spring N timings increased GPC. The NUE (range: 20–311 kg kg‐1) was inversely related to N availability, and Fall N timing had the lowest NUE. Our results highlighted physiological reasons for genotype‐specific GPC performance and suggested agronomic opportunities to simultaneously improve wheat yield and GPC.