We describe a high-resolution cytogenetic map for 46 DNA markers previously assigned to the short arm of human chromosome 1. Using fluorescence in situ hybridization on simultaneously R-banded prometaphase chromosomes, a refined map position was found for 45 probes. For 6 of these probes, additional hybridization sites were observed and for another 7 probes, conflicting results were found with regard to previous localizations. For some probes with overlapping map positions, probe order could be determined by dual-color hybridization on elongated chromosomes. The present high-resolution map can be used to refine the previously published composite map and also provides additional landmarks for the construction of a contig map of the short arm of chromosomes 1.