MAD2 (mitotic arrest deficient 2) is a highly conserved protein involving in spindle checkpoint control. MAD2 locates at spindle-unattached kinetochores during prophase and dissolves from spindle-attached kinetochores towards metaphase. In this study, we isolated homologous genes encoding for MAD2 from hexaploid wheat. The three homoeologous genes on the long arms of the group-2 chromosomes shared approximately 99% similarity of the nucleotide sequence in coding regions. The intron-exon structures of the three homoeologues were also conserved, showing high similarities to that of the Arabidopsis MAD2 gene. All three homoeologues were transcribed in roots and spikes but not in leaves. We generated antibodies against the polypeptides with amino acid sequences derived from the cDNA sequences of the wheat MAD2 homologues. Using these antibodies, we found MAD2 in wheat root-tip cells to change in location and amount through the cell cycle, similar to those reported for human MAD2. Intense immunostaining signals were observed at the centromeres of all metaphase chromosomes when root-tips were treated with colchicine, a microtubule-destabilizing drug, but no signals were observed in untreated chromosomes. Thus, the wheat MAD2 protein could be a good marker for the functional kinetochores of metaphase chromosomes in wheat.