Measurement of biochemical markers allows the quantification of wheat (Triticum spp.) grain tissue proportions in milling fractions. In order to evaluate the ability of extending this methodology to an unknown wheat grain batch, the variability of the markers in the different tissues was assessed on various wheat cultivars. Ferulic acid trimer amounts in the outer pericarp ranged from 0.97 to 1.67 mg mg_1 (dm) with an average value equal to 1.31 mg mg_1 (dm). Alkylresorcinols amounts in a composite layer, including the testa, the inner pericarp and the nucellar epidermis, ranged from 10.5 to 16.7 mg g_1 (dm), with an average value equal to 14.0 mg g_1 (dm). In the aleurone layer, phytic acid amounts ranged from 94.9 to 187.2 mg g_1 (dm) with an average value equal to 152 mg g_1 (dm) whereas, para-coumaric acid ranged from 0.08 to 0.29 mg mg_1 with an average level of 0.18 mg mg_1. In the embryonic axis, wheat germ agglutinin ranged from 879 mg g_1 to 2086 mg g_1 with an average value of 1487 mg g_1. The impact of this variability on tissue proportion determination was evaluated and a strategy to decrease the prediction error was suggested. Percentages of the outer pericarp, intermediate layer (including the testa), aleurone layer and embryonic axis within grains were calculated and their variability discussed