Glucosinolates (GLS) are secondary metabolites commonly occurring in Brassica crops and more than 130 different GLS have been reported in diverse plants. Recent studies have indicated that isothiocyanate (ITC) derived from GLS by hydrolysis had a potential for anticancer activity against several rumor cells on human. In addition, it was found that glucoraphenin (GRE) and glucoraphasatin (GRH) were abundant and differently regulated in radish plant, depending upon organs and developmental stages. Microspores isolated from flower buds of radish were cultured in vitro to obtain doubled haploid (DH; but homozygous) lines in a short time period. The present study was conducted to determine the concentration of GRE and GRH, an immediate precursor of ITC from DH lines of radish plant. Total 41 DH lines were selected based on flow cytometry analysis. The seeds, obtained by bud pollination from the DH lines, were planted and 3-weeks-old young seedlings were used for the major aliphatic GLS analysis. Amounts of GRH were highly variable from the DH lines ranging from 2.3 to 31.5 mg·g-1 dry weight (DW). The donor plant (DP) contained 18.4 mg·g-1 DW. It was noticed that there were 6-fold differences in the amounts of GRE between the highest and lowest DH lines. Among 41 lines tested, 14 DH lines of radish plant were significantly reduced in the amount of sum of GRH and GRE compared those of the donor plant (P<0.05), whereas only three lines increased. The results obtained in the present study will lend to select genotypes with low and high GLS contents of radish plant. In addition, those DH lines will aid to elucidate a biosynthetic pathway of the aliphatic GLS in radish plant, which remain for the most part unsolved.