A laboratory protocol was developed to assess glucose and ethanol yields from wheat. The impact of the analyzed wholemeal flour quantity and the saccharification on the amount of released glucose was estimated. The whole process including the analytical methods (glucose and ethanol) was repeatable and reproducible. This protocol was used to assess the glucose and ethanol yields of six varieties and of a complete set of hexaploid near-isogenic waxy lines of cv. Trémie grown in three locations. As compared to the normal line of Trémie, double null (AnBnD) and triple null (nAnBnD) isogenic lines showed a low Hagberg falling number (218, 65, and 63 s, respectively), a higher grain protein content (10.7, 11.5, and 12.1% DM, respectively), a lower glucose yield (728, 703, and 707 kg/t, respectively), and a lower ethanol yield (463, 453, and 452 L/t, respectively). These values indicate a strong involvement of alleles encoded at Wx-B1 and Wx-D1 loci in grain composition.