Barley (Hordeum vulgare L.) is an important cereal crop and a model species for Triticeae genomics. To lay the foundation for hierarchical map-based sequencing, a genome-wide physical map of its large and complex 5.1 billion base pair genome was constructed by high-information content fingerprinting of almost 600,000 bacterial artificial chromosomes (BACs) representing 14x haploid genome coverage. The resultant physical map comprises 9,265 contigs with a cumulative size of 4.9 Gb representing 96 % of the physical length of the barley genome. The reliability of the map was verified through extensive genetic marker information and the analysis of topological networks of clone overlaps. A minimum tiling path of 66,772 minimally overlapping clones was defined that will serve as template for hierarchical clone-by-clone map-based shotgun sequencing. We integrated whole-genome shotgun sequence (WGS) data from the individuals of two mapping populations with published BAC survey sequence information to genetically anchor the physical map. This novel approach in combination with the comprehensive WGS datasets allowed us to independently validate and improve a previously reported physical and genetic framework. The resources developed in this study will underpin fine-mapping and cloning of agronomically important genes and the assembly of a draft genome sequence.