A high-resolution cytogenetic map of human chromosome 9 was constructed with 203 newly isolated cosmid markers by direct R-banding fluorescence in situ hybridization. The clones were localized preferentially to R-positive bands throughout chromosome 9. Although the number of clones was roughly proportional to the R-band size, many more clones (ca. 80) were mapped at the q34 region compared to its physical size. Based on these cytogenetic mapping data, one can establish physical contig maps with cosmids and yeast artificial chromosomes and a genetic linkage map essential for positional cloning of responsible genes.