In order to generate a physical map of the Arabidopsis thaliana genome based on bacterial artificial chromosome clones (BACs), an iterative high throughput hybridisation strategy was applied and its efficiency was evaluated. Thus, probes generated from both ends of 500 BAC clones selected from the Arabidopsis-IGF-BAC library were hybridised to the entire library gridded on high density filters. The 1000 hybridisation reactions identified 4496 clones (41.8% of the complete library, or 50.3% if organellar, centromeric, and ribosomal DNA carrying clones are excluded) which were assembled into a minimum of 220 contigs. These results demonstrate the viability of the applied 'double-end clone-limited/sampling without replacement' hybridisation strategy for the generation of a high resolution physical map, and provide a highly useful resource for map-based gene cloning approaches and further genome analysis.