Cytoplasmic male sterility caused by DCGMS (Dongbu cytoplasmic and genic male-sterility) cytoplasm and its nuclear restorer-of-fertility locus (Rfd1) with a linked molecular marker (A137) have been reported in radish (Raphanus sativus L.). To construct a linkage map of the Rfd1 locus, linked amplified fragment length polymorphism (AFLP) markers were screened using bulked segregant analysis. A 220-bp linked AFLP fragment sequence from radish showed homology with an Arabidopsis coding sequence. Using this Arabidopsis gene sequence, a simple PCR marker (A220) was developed. The A137 and A220 markers flanked the Rfd1 locus. Two homologous Arabidopsis genes with both marker sequences were positioned on Arabidopsis chromosome 3 with an interval of 2.4 Mb. To integrate the Rfd1 locus into a previously reported expressed sequence tag (EST)-simple sequence repeat (SSR) linkage map, the radish EST sequences located in three syntenic blocks within the 2.4-Mb interval were used to develop single nucleotide polymorphism (SNP) markers for tagging each block. The SNP marker in linkage group 2 co-segregated with male fertility in an F2 population. Using radish ESTs positioned in linkage group 2, five intron length polymorphism (ILP) markers and one cleaved amplified polymorphic sequence (CAPS) marker were developed and used to construct a linkage map of the Rfd1 locus. Two closely-linked markers delimited the Rfd1 locus within a 985-kb interval of Arabidopsis chromosome 3. Synteny between the radish and Arabidopsis genomes in the 985-kbp interval were used to develop three ILP and three CAPS markers. Two ILP markers further delimited the Rfd1 locus to a 220-kb interval of Arabidopsis chromosome 3.