The quantitative trait loci (QTLs) on chromosomes 7BL and 3BS from Halberd have been used as a major source of tolerance to late maturity α amylase (LMA) within Australian wheat breeding programs. New simple sequence repeat (SSR) markers identified from the sequencing of Bacterial Artificial Chromosome (BAC) clones from the wheat cv. Renan library, and known SSRs, were used to characterise these major QTLs. The reduction or elimination of the LMA defect in wheat cultivars is a major goal for wheat breeding programs and is confounded by the complexity in measuring the trait unambiguously. In this haplotyping study focussing on two significant chromosomal regions, markers and combinations of markers were investigated for their ability to discriminate between 39 Australian and Mexican wheat lines differing in levels of LMA. Genetic relationships among these wheat lines estimated by cluster analysis of molecular marker data were combined with phenotypic information in order to calibrate the genotypes of the wheat lines against their LMA phenotype. It was evident that some SSRs from the respective QTLs had greater discriminating power than others to identify LMA phenotypes. Discrimination was not, however, absolute and a statistical analysis of the data defined a risk factor associated with particular combinations of alleles, for use in early selection or backcrossing.