Powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is one of the most serious diseases in wheat. DNA markers such as AFLP and SSR have been very effective for defining and mapping new resistance genes in plants. M53 (YAV2/TEZ//Ae. squarrosa 249) is a synthetic wheat of Triticum durum and Aegilops tauschii. This synthetic wheat carries a resistance gene, tentatively designated Pm-M53, conferring resistance to the No.15 isolate of Bgt that has been prevailing in wheat fields in Beijing area and endangering the local wheat production in recent years. In the present study, the resistance pattern of a F2 population (118 individuals) derived from cross of M53 and Wan7107 was analyzed through inoculation with No.15 isolate. The response patterns showed that the R∶S segregation ratio fits 3∶1, suggesting that the gene of interest be a single dominant gene, which was confirmed by F3 progeny test. AFLP and SSR markers were then used to search genetically linked markers to the target gene. Using JoinMap V3.0 with Kosambi’s function and other options left at default values, two AFLP markers P16M16-109 (Apm109) and P5M16-161 (Apm161) were identified to be tightly linked to Pm-M53, with genetic distances of 1.0 and 3.0 cM, respectively, and three SSR markers, Xwmc289b, Xgwm583 and Xgwm292, all assigned to the long arm of chromosome 5D, were also found to be associated with the gene, with genetic distances of 20.0, 33.0 and 24.0 cM, respectively. These markers flanked Pm-M53. Apm161, Xwmc289b and Xgwm583 were placed at one side, Apm109 and Xgwm292 at the opposite. From proximal to distal in order, the arrangement of the gene and its coupling markers on the long arm of chromosome 5D was Xgwm583, Xwmc289b, Apm161, Pm-M53 Apm109 and Xgwm292. Physical location of the gene was carried out using two Chinese Spring nullisomic-tetrasomic lines CSN5BT5D and CSN5DT5B, and two ditelosomic lines CSDT5DS and CSDT5DL by means of tightly linked AFLP marker APm109. The results therefore corroborate the allocation of the gene to chromosome arm 5DL. The specific position on the chromosome indicated Pm-M53 was a new powdery mildew resistance gene. It could play an important role in wheat breeding programs for powdery mildew resistance. The relationship of the gene with other reported resistant genes to powdery mildew on the D genome was discussed.