Barley yellow dwarf virus (BYDV) can cause significant losses of wheat worldwide. The long arm segment of Thinopyrum intermedium chromosome 7Ai#1 carrying the BYDV resistance gene Bdv2 was translocated to the distal region of the long arm of wheat chromosome 7D in translocation line Yw642. In this study, 40 wheat EST sequences located in the distal region of 7DL were explored to identify specific PCR markers for the Bdv2 region on the basis of the homoeologous relationship between wheat chromosome 7D and Th.intermedium chromosome 7Ai#1. Our results revealed 8 novel EST-PCR markers specific to the Bdv2 region, including 5 EST-STS markers of BE404744, BE498985, BE591497, BG606695 and BQ161842, and 3 EST-SSCP markers of BE404953, BG312663 and BE498985. These EST-PCR markers could distinguish Bdv2 from another BYDV-resistance gene located on Th.intermedium chromosome 2Ai-2. These specific bands for the Bdv2 region were further cloned and sequenced. The sequencing analysis indicated that the specific sequences for the Bdv2 region were highly homologous with the original wheat EST sequences that were used to design primers, and encode respectively a protein kinase, P450, centrin, transducin, and a hypothetical protein. This study created a starting point for eventual cloning of the Bdv2 gene and understanding the defense mechanism.