Key message <p>Survey of tocopherol and identification of genetic loci in wheat grain: toward a better understanding for breeding tocopherol cultivars.</p> Abstract <p>Tocopherol is essential in maintaining human sex hormone as well as antioxidation and immune functions. In this study, the contents of α-, γ- and δ-tocopherol in a Chinese wheat mini-core collection were measured with high-performance liquid chromatography. The effects of various factors such as accession types, winter/spring types and grain colors on tocopherol content were analyzed, and the genetic loci for tocopherol were identified with genome-wide association analysis and linkage analysis. There was no significant difference in total tocopherol in grain of wheat grown in three environments. Seven high-tocopherol (≥ 110&#xa0;μg/g) germplasms were selected. The genotypic and environmental effects on total tocopherol and its three components ranked G &gt; G × E &gt; E and the tocopherol content were not correlated with accession types, spring/winter variety, red/white grain and release years. Among the eleven agronomic and four grain traits, α-tocopherol was positively correlated with grain thickness, γ-tocopherol was positively correlated with heading date, maturity date and flag leaf length, and γ-, δ- and total tocopherol were negatively correlated with the number of kernels in the middle of the spike. The total tocopherol showed positive correlation with lycopene and lutein. A total of twenty-seven QTL associated with tocopherol were identified with genome-wide association analysis, with obvious additive effects. The fine mapping of <i>Qδ.toc.6 A</i> narrowed functional region down to a 5.26-Mb region via analyzing the tocopherol content among genotypes in secondary mapping population. We proposed five candidate genes with known pathways for tocopherol synthesis in wheat, potentially useful in breeding high-tocopherol wheat varieties.</p>