The combined application of nitrogen (N) and zinc (Zn) fertilizers is a promising agronomic strategy for the biofortification of wheat grain with Zn for human nutrition. A glasshouse experiment was carried out to assess the effects of supplying N on the uptake, translocation and accumulation of Zn in tissues of two wheat genotypes (Quartzo and BRS Parrudo) with contrasting potential for grain Zn biofortification. Winter wheat genotypes were grown to maturity in 5 cm diameter, 100 cm length tubes filled with a mixture of sand, grit and gravel (40:40:20 v/v/v) over a layer of 0.1 m3 of gravel, and supplied a full nutrient solution with low Zn (0.15 μM) or high Zn (2.25 μM) and low N (0.4 mM) or high N (4.0 mM) concentrations. High N supply increased biomass production, Zn concentration and Zn content of straw and grain in both Quartzo and BRS Parrudo. Grain Zn content more than doubled when the supplies of Zn and N were both increased from low to high in both genotypes. Quartzo had a greater grain yield than BRS Parrudo. BRS Parrudo had greater grain Zn concentration and Zn content than Quartzo. A greater N supply promoted better uptake, translocation to the shoot and accumulation of Zn within the grain. Quartzo and BRS Parrudo differed in their partitioning of biomass and Zn between tissues. It might be possible to combine the greater grain yield of Quartzo with the greater grain Zn accumulation of BRS Parrudo to deliver a greatly improved genotype for human food security.