Sequence-tagged sites (STSs) derived from end fragments of chromosome-specific yeast artificial chromosomes (YACs) can facilitate the assembly of an overlapping YAC/STS map. Contigs form rapidly by iteratively screening YAC collections with end-fragment STSs from YACs that have not yet been detected by any previous STS. The map is rendered rapidly useful during its assembly by incorporating supplementary STSs from genes and genetic linkage probes with known locations. Methods for the systematic development and testing of the end-fragment STSs are given here, and a group of 100 STSs is presented for the X chromosome. The mapping strategy is shown to be successful in simulations with portions of the X chromosome already largely mapped into overlapping YACs by other means.