A powdery mildew resistance gene MlHLT derived from a Chinese wheat landrace maps within a 3.6 centimorgan (cM) genetic interval spanning a 13.4 megabase (Mb) physical genomic region on chromosome 1DS. Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt) is a devastating disease that can cause severe yield losses. Chinese wheat landrace Hulutou confers nearly immune resistance against prevailing Bgt isolate E09 in Beijing. Genetic analysis indicate that the powdery mildew resistance of Hulutou is controlled by a single dominant gene, provisionally designated MlHLT. Bulked segregant analysis(BSA) and simple sequence repeat (SSR) mapping showed that MlHLT is located on chromosome arm 1DS between markers Xgwm337 and Xcfd83/Xcfd72. By applying comparative genomics analysis, collinearity genomic regions of the MlHLT locus on Aegilops tauschii chromosome 1DS were identified in Brachypodium distachyon chromosome 2, rice chromosome 5 and sorghum chromosome 9, respectively. Three new polymorphic markers were developed using the draft genome sequences and the extended single nucleotide polymorphism (SNP) marker sequences of Ae. tauschii accession AL8/78, as well as the Triticum aestivum cv. Chinese Spring 454 contig sequences and the International Wheat Genome Sequencing Consortium (IWGSC) survey sequences. MlHLT mapped into a 3.6 cM genetic interval spanning 13.4 Mb physical genomic region containing seven contigs (ctg220, ctg4623, ctg1063, ctg5929, ctg3163, ctg699 and ctg1065) on 1DS that has synteny with a 369.8 kb genomic region in Brachypodium, a 380.8 kb genomic region in rice and a 298.4 kb genomic region in sorghum. The genetic and physical maps of MlHLT provide framework for map-based cloning and marker-assisted selection (MAS) of the powdery mildew resistance gene MlHLT in Hulutou.