Powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is one of many severe diseases that threaten bread wheat (Triticum aestivum L.) yield and quality worldwide. The discovery and deployment of powdery mildew resistance genes (Pm) can prevent this disease epidemic in wheat. In a previous study, we transferred the powdery mildew resistance gene Pm57 from Aegilops searsii into common wheat and cytogenetically mapped the gene in a chromosome region with the fraction length (FL) 0.75-0.87, which represents 12% segment of the long arm of chromosome 2Ss#1. In this study, we performed RNA-seq using RNA extracted from leaf samples of three infected and mock-infected wheat-Ae. searsii 2Ss#1 introgression lines at 0, 12, 24, and 48 h after inoculation with Bgt isolates. Then we designed 79 molecular markers based on transcriptome sequences and physically mapped them to Ae. searsii chromosome 2Ss#1- in seven intervals. We used these markers to identify 46 wheat-Ae. searsii 2Ss#1 recombinants induced by ph1b, a deletion mutant of pairing homologous (Ph) genes. After analyzing the 46 ph1b-induced 2Ss#1L recombinants in the region where Pm57 is located with different Bgt-responses, we physically mapped Pm57 gene on the long arm of 2Ss#1 in a 5.13 Mb genomic region, which was flanked by markers X67593 (773.72 Mb) and X62492 (778.85 Mb). By comparative synteny analysis of the corresponding region on chromosome 2B in Chinese Spring (T. aestivum L.) with other model species, we identified ten genes that are putative plant defense-related (R) genes which includes six coiled-coil nucleotide-binding site-leucine-rich repeat (CNL), three nucleotide-binding site-leucine-rich repeat (NL) and a leucine-rich receptor-like repeat (RLP) encoding proteins. This study will lay a foundation for cloning of Pm57, and benefit the understanding of interactions between resistance genes of wheat and powdery mildew pathogens.