The intervals containing two major quantitative trait loci (QTL) from a Spanish barley landrace conferring broad spectrum resistance to Blumeria graminis were subjected to marker saturation. First, all the available information on recently developed marker resources for barley was exploited. Then, a comparative genomic analysis of the QTL regions with other sequenced grass model species was performed. As a result of the first step, 32 new markers were added to the previous map and new flanking markers closer to both QTL were identified. Next, syntenic integration revealed that the barley target regions showed homology with regions on chromosome 6 of rice (Oryza sativa), chromosome 10 of Sorghum bicolor and chromosome 1 of Brachypodium distachyon. A nested insertion of ancestral syntenic blocks on Brachypodium chromosome 1 was confirmed. Based on sequence information of the most likely candidate orthologous genes, 23 new barley unigene-derived markers were developed and mapped within the barley target regions. The assessment of colinearity revealed an inversion on chromosome 7HL of barley compared to the other three grass species, and nearly perfect colinearity on chromosome 7HS. This two-step marker enrichment allowed for the refinement of the two QTL into much smaller intervals. Inspection of all predicted proteins for the barley unigenes identified within the QTL intervals did not reveal the presence of resistance gene candidates. This study demonstrates the usefulness of sequenced genomes for fine mapping and paves the way for the use of these two loci in barley breeding programs.