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Waxy (Wx) protein is a key enzyme for synthesis of amylose in endosperm. Amylose content in wheat grain influences the quality of end-use products. Seven alleles have been described at the Wx-D1 locus, but only two of them (Wx-D1b, Wx-D1e) were genotyped with codominant markers. The waxy wheat line K107Wx1 developed by treating ‘Kanto 107’ seeds with ethyl methanesulphonate carries the Wx-D1d allele. However, no molecular basis supports this nomenclature. In the present study, DNA sequence analysis confirmed that a single nucleotide polymorphism in the sixth exon of Wx-D1 changed tryptophan at position 301 into a termination codon. Based on this sequence variation, a PCR-based KASP marker was developed to detect this point mutation using 68 BC8F1 plants and 297 BC8F2 lines derived from the cross ‘Ningmai 14’*9/K107Wx1. Combined with codominant markers for the Wx-A1 and Wx-B1 alleles, waxy and non-waxy near-isogenic lines were distinguished. The KASP marker was efficient in identifying the mutant allele and can be used to transfer waxiness to elite lines.