Genetic analysis and gene mapping of the 4-methylthio-3-butenyl glucosinolate-less trait of white radish were performed and a white radish cultivar with new glucosinolate composition was developed. A spontaneous mutant having significantly low 4-methylthio-3-butenyl glucosinolate (4MTB-GSL) content was identified from a landrace of Japanese white radish (Raphanus sativus L.) through intensive evaluation of glucosinolate profiles of 632 lines including genetic resources and commercial cultivars using high-performance liquid chromatography (HPLC) analysis. A line lacking 4MTB-GSL was developed using the selected mutant as a gene source. Genetic analyses of F1, F2, and BC1F1 populations of this line suggested that the 4MTB-GSL-less trait is controlled by a single recessive allele. Using SNP and SCAR markers, 96 F2 plants were genotyped, and a linkage map having nine linkage groups with a total map distance of 808.3 cM was constructed. A gene responsible for the 4MTB-GSL-less trait was mapped between CL1753 and CL5895 at the end of linkage group 1. The genetic distance between these markers was 4.2 cM. By selfing and selection of plants lacking 4MTB-GSL, a new cultivar, 'Daikon parental line No. 5', was successfully developed. This cultivar was characterized by glucoerucin, which accounted for more than 90% of the total glucosinolates (GSLs). The total GSL content in roots was ca. 12 μmol/g DW, significantly lower than those in common white radish cultivars. Significance of this line in radish breeding is discussed.