High-resolution physical maps can be used as a scaffold for several subsequent studies, such as sequencing projects and positional cloning of disease genes and genetic elements that regulate gene expression. Here we describe a method for fast, high-resolution physical mapping on stretched DNA molecules, based on a combinatorial multi-FISH approach. Fluorescent labels are assigned to a binary code and probes are identified by a binary tag according to their labeling. To validate the approach, we have mapped eight probes covering a region of about 300 kb on human chromosome 11 with three hybridization assays. This approach enables one to determine the structural organization of a large region by means of the order of its clones, without ambiguities. The structure established in a control cell constitutes a reference for further studies, to detect rearrangements displayed by disease cells and to find differences shown by different cell types and organisms.