The general usefulness of fluorescence in situ hybridization (FISH) for the physical mapping of the human genome is greatly enhanced by improved DNA resolution. Several techniques have been described for decondensing or stretching interphase chromatin in a linear fashion, allowing long-range FISH mapping in finer detail. Highly extended linear chromatin can be hybridized over physical distances of at least several megabases, possibly over the whole length of a chromosome. By multi-color FISH, we have determined the order and overlaps of YACs from a 5q34 - 35 contig. Cosmids can be localized within larger YAC clones. Extended chromatin mapping can be applied as an adjunct ordering technique in genome studies.