Dasypyrum villosum, a wheat wild relative, carries many disease resistance genes which have not been cloned yet. Considering majority of the plant disease resistance (R) proteins belong to the nucleotide-binding-leucine rich repeat (NBS-LRR) -containing R gene family, the conserved sequences of NBS-LRR resistance genes were used to design a primer pair MLA6-1-F/MLA6-1-R in present study. Genomic DNA and cDNA from Dasypyrum villosum were amplified with the primer pair, and in total twenty-three sequences were achieved. They were roughly divided into two groups based on their similarity in sequences with each other aligned by DNAMAN soft and with the resistance genes searched by NCBI BLAST. In Mla/RGAs-like group, twelve sequences had similarities of 91–93% and 87–90% to Aegilops tauschii resistance gene analogy (RGAs) and Triticum monococcum DV92 Mla1 gene, respectively. Another sequence H6 had 85% identity with Hordeum vulgare genes Mla1, Mla6, Mla12. According to the five sequences of the resistance gene analogues (RGAs) H-56/d6, H-66/b2 and CDS40, in which the sequences from both gDNA and cDNA were all obtained, primer pairs were redesigned to amplify common wheat, Dasypyrum villosum, Triticum durum- Dasypyrum villosum amphidiploid and its hybrids, and the wheat lines simultaneously carrying certain Dasypyrum villosum chromosome arms or entire chromosome such as 1VL and 2VS, 2VS and 4V, or 6V, separately. The result showed that the primer pair of H-56/d6 could not amplify an expected fragment from the lines with 1VL, 2VS, 4V and 6V, and primer pair derived from sequence of H-66/b2 could get amplification from Dasypyrum villosum, Triticum durum- Dasypyrum villosum amphidiploid, its hybrids, and the wheat lines carrying Dasypyrum villosum chromosome 1VL and 6VL, but not from the lines with 2VS, 4V and 6VS. While, primer pair designed from the sequence of CDS40 could not amplify an expected fragment specifically from the 6VS translocation line 10SR3109 and Pm97033. The markers not only confer novel molecular markers for tracing the alien chromosome in wheat background, but can also be tightly linked to R gene and R genes clusters, which will lay a foundation for further isolation and identification of the resistance genes from Dasypyrum villosum.