Second-generation sequencing now provides the potential for low-cost generation of whole-genome sequences. However, for large-genome organisms with high repetitive DNA content, genome-wide short read sequence assembly is currently impossible, with accurate ordering and localization of genes still relying heavily on integration with physical and genetic maps. To facilitate this process, we have used Agilent microarrays to simultaneously address thousands of gene sequences to individual BAC clones and contiguous sequences that form part of an emerging physical map of the large and currently unsequenced 5.3-Gb barley genome. The approach represents a cost-effective, highly parallel alternative to traditional addressing methods. By coupling the gene-to-BAC address data with gene-based molecular markers, thousands of BACs can be anchored directly to the genetic map, thereby generating a framework for orientating and ordering genes, and providing direct links to phenotypic traits.