Wheat Q gene located in the homoeologous group 5 chromosomes is extraordinarily important for the widespread cultivation of wheat, because of its effects on many domestication-related morphological traits. The full length of Q gene from each locus of 5A, 5B and 5D is 3.2-3.4 kb, which makes it difficult to verify them from each other by PCR and electrophoresis. This study focused on developing molecular makers to verify the three different wheat Q gene copies from homoeologous chromosomes 5A, 5B and 5D. The locus-specific markers based on the INDELs in the 9th intron region of Q gene were designed to verify the three homoeologous loci from each other. Three gene segments of Gene Q were cloned from different wheat materials as molecular markers. These three makers of 536 bp, 665 bp and 578 bp were identified from each homoeologous loci of 5A, 5B and 5D. These markers are useful for tracing different loci of Q gene and can also be used in the identification of genetic materials involved in homoeologous group 5 chromosomes.