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A molecular marker associated with male fertility restoration was newly identified in onion in this study using random amplification of polymorphic DNA (RAPD) and bulk segregant analysis. A polymorphic amplicon from RAPD reaction was observed in DNA from fertile plants. The amplicon was designated as the RAPD marker OBC14.1000 and a 980-base pair (bp) nucleotide sequence was obtained. Genome walking was performed to obtain the flanking sequences of the OBC14.1000 marker to develop a co-dominant marker. The acquired sequences were a 2049-bp fragment linked to a male-fertile allele and a 1621-bp fragment linked to a male-sterile allele. Approximately 95% similarity was shown when the nucleotide sequences between fertile and sterile alleles were aligned. Using a polymorphic region containing a restriction site of Ava II in a fertile allele, the RAPD marker OBC14.1000 was successfully converted into a co-dominant CAPS marker and it was designated as ACms.1100. It appeared to be positioned much closer to the restorer-of-fertility locus, Ms , based on genotyping analysis as compared to other currently available markers. No putative homology was found to the newly identified ACms.1100 marker sequences through a Blast database search and the identification of sequences is still unknown.