This chapter describes a nonradioactive, agarose gel-based, high-throughput DNA restriction digest fingerprinting methodology first described by Marra et al. (1) for use in the construction of high-resolution physical maps from low-copynumber, large-insert clones. The procedure is robust and allows for the recovery of clone insert size information. Initially used to construct sequence tag site (STS)-based contigs (1), the methodology has also been applied to whole-genome, random-clone strategies that have resulted in the construction of high-resolution, sequence-ready physical maps of the genomes of Arabidopsis thaliana (2,3), human (4,5), Caenorhabditis briggsae (6), and Cryptococcus neoformans (7). The methodology is currently being employed in the construction of physical maps for several other large, mammalian genomes, such as those of mouse (8), rat and bovine.