Error-prone repair of radiation-induced DNA double-strand breaks (DSBs) results in DNA mutation that is essential for mutation breeding. Non-homologous end joining might be the principal DSB repair mechanism in eukaryotes, which is mediated and activated by Ku protein, a heterodimer of 70 and 80 kDa subunits. In this study, on the basis of complementary DNA (cDNA), the genomic sequences of TaKu70 and TaKu80 genes in all the three genomes of wheat were characterized. Only single-nucleotide substitutions and no insertions or deletions were detected in the exons of TaKu70 and TaKu80 genes. The size of the introns exhibited a slight variation between the sequences. Yeast two-hybrid analysis demonstrated that TaKu70 and TaKu80 formed a heterodimer, and electrophoretic mobility shift assays revealed that this heterodimer bound to double-stranded DNA, but not to single-stranded DNA. The quantitative polymerase chain reaction analysis revealed that the expression of TaKu70 and TaKu80 genes was up-regulated under γ-ray irradiation in a dose-dependent manner in the seedlings of wheat. These results suggest that TaKu70 and TaKu80 form a functional heterodimer and are associated with the repair of the induced DSBs in wheat.