Human zinc (Zn) deficiency is a worldwide problem, especially in developing countries due to the prevalence of cereals in the diet. Among different alleviation strategies, genetic Zn biofortification is considered a sustainable approach. However, it may depend on Zn availability from soils. We grew Zincol-16 (genetically-Zn-biofortified wheat) and Faisalabad-08 (widely grown standard wheat) in pots with (8 mg kg−1) or without Zn application. The cultivars were grown in a low-Zn calcareous soil. The grain yield of both cultivars was significantly (P≤0.05) increased with that without Zn application. As compared to Faisalabad-08, Zincol-16 had 23 and 41% more grain Zn concentration respectively at control and applied rate of Zn. Faisalabad-08 accumulated about 18% more grain Zn concentration with Zn than Zincol-16 without Zn application. A near target level of grain Zn concentration (36 mg kg−1) was achieved in Zincol-16 only with Zn fertilisation. Over all, the findings clearly signify the importance of agronomic Zn biofortification of genetically Zn-biofortified wheat grown on a low-Zn calcareous soil.