The waxy gene (Wx-1) encodes the granule-bound starch synthase (GBSSI or waxy protein, Wx), which is the key enzyme that catalyzes amylose synthesis in wheat. In this study, tetraploid wheat (Triticum turgidum durum L.) was mutagenized by ethyl methanesulfonate, and M2 populations of 3474 plants were generated. The Wx-A1 orWx-B1 null mutants were identiﬁed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The results of cloning showed that there was a single nucleotide polymorphism (SNP) of G1086A in Wx-A1 of line M2-219, which is predicted to generate a stop codon, thereby resulting in premature termination of the WxA1 protein. Two SNPs, G207A and G1122A, weredetected in Wx-B1 of M2-504; the former results in the amino acid substitution from Serine to Asparagine, whereas the latter disrupts the production of the WxB1 protein. The Wx-A1 and the Wx-B1 null mutants were crossed; the lines with variable waxy proteins compositions were characterized. The loss of function involving the waxy protein has resulted in changes in starch properties in various lines.The line lacking both Wx-A1 and Wx-B1 had the lowest amylose content and highest onset and peak gelatinization temperature; the lack of Wx-B1 results in an increase in amylose content instead of loss of Wx-A1. In addition, the abundance of B-type starch granules signiﬁcantly increased in the Wx null line. Because waxy proteins largely inﬂuence the ﬂour quality, then the Waxy lines with lower amylose content would be useful in wheat quality breeding.