For efficient introgression of the downy mildew resistance gene from a resistant cultivar into domestic breeding lines, molecular markers used for marker-assisted backcrossing (MAB) were developed in onion (Allium cepa L.). The resistance gene (Pd) was originally introgressed from a wild species, A. roylei, by interspecific hybridization, and the resistant gene was known to be positioned at the end of chromosome 3. Therefore, cDNA sequences of loci located at the ends of chromosome 3 of two linkage maps were obtained from a transcriptome database. Primer pairs were designed on exon sequences of eight loci. Among them, the PCR products of the i25255 locus showed length polymorphism between A. roylei and onions, and both large and small-sized PCR products were observed in the resistant cultivar. Sequence analysis showed that a 67-bp indel existed in the intron sequences. Based on this indel polymorphism, a simple PCR marker, designated DMR1, was developed. Analysis of diverse onion accessions showed that no accessions contained the A. roylei-specific marker genotype except for the resistant cultivar. These results indicated that the DMR1 marker was successfully tagging the A. roylei fragment harboring the downy mildew resistance gene, and the resistant cultivar was heterozygous for the resistance gene. After further analysis of multiple loci positioned at chromosome 3, a range of the A. roylei fragment introgressed in the resistant cultivar was determined in two linkage maps. On the basis of the range of the A. roylei fragment, three molecular markers used for recombinant selection in MAB were also developed.