Since few genetic markers are available in bunching onion, many DNA markers are needed for the construction of a primary basis for the breeding of this crop. We report here the development of microsatellite markers in bunching onion. A size-fractionated genomic library was constructed from genomic DNA of bunching onion cv. ‘Kujo Futo’ and screened with a mixture of (GA)₁₅ and (GT)₁₅ oligonucleotide probes. From approximately 180,000 clones we isolated and sequenced 94 positive clones. A total of 52 clones were identified as having microsatellite repeats. Of them 49 had a GT motif, while only one had a GA motif, which is known to be more frequent than GT in most plant species. This is the first report of the abundance of GT over GA in Allium spp. Of 50 specific PCR primer pairs designed for the microsatellite-containing clones, 33 primer pairs amplified polymorphic loci in nine cultivars of bunching onion, 115 alleles detected in total. These results indicate that microsatellites with dinucleotide motifs, especially with GT, are promising sources of highly informative genetic markers in bunching onion.