Onion (Allium cepa L.; 1C=15,000 Mb) is an agriculturally important plant. The genome of onion has been extensively studied at the conventional cytogenetic level, but molecular analyses have lagged behind due to its large genome size. To overcome this bottleneck, a partial bacterial artificial chromosome (BAC) library of onion was constructed. The average insert size of the BAC library was about 100 kb. A total of 48,000 clones, corresponding to 0.32 genome equivalent, were obtained. Fluorescent in situ hybridization (FISH) screening resulted in identification of BAC clones localized on centromeric, telomeric, or several limited interstitial chromosomal regions, although most of the clones hybridized with entire chromosomes. The partial BAC library proved to be a useful resource for molecular cytogenetic studies of onion, and should be useful for further mapping and sequencing studies of important genes of this plant. BAC FISH screening is a powerful method for identification of molecular cytogenetic markers in large-genome plants.