An enzyme-linked immunosorbent assay (ELISA) for the discrimination of Wx-B1a and Wx-B1b genotypes at the granule-bound starch synthase I (GBSSI) or waxy locus of hexaploid wheat ( Triticum aestivum L.) was adapted to a high-throughput, 96-well microtitre plate format. This test is applicable to the direct analysis of starch, flour, or crushed grain and requires less than 1 grain to perform. Several hundred samples may be routinely analysed in one day. The assay was validated using quantitative trait locus (QTL) analysis of a doubled haploid mapping population of the cross Cranbrook ( Wx-B1a )/Halberd ( Wx-B1b). This demonstrated that the assay unambiguously identified 153 of 161 lines analysed, with a highly significant QTL (LRS value 270) accounting for 83% of ELISA variation, at the Wx-B1 locus on chromosome 4AL. In addition, measurement of total GBSSI variation using a non-isoform-specific GBSSI detection monoclonal antibody also gave a significant QTL (LRS of 84, accounting for 42% of ELISA variation) at the Wx-B1 locus. Application of the assay to crude flour extracts of 8 grains for each of 1093 progeny from 4 crosses segregating at the Wx-B1 locus permitted the unambiguous scoring of lines as pure Wx-B1a or pure Wx-B1b . The scoring by ELISA was strongly related to the flour swelling volume of the lines, thus demonstrating the utility of this high-throughput screening method for the faster, more efficient development of Australian noodle wheats.