Glucosinolates (GSLs), beneficial secondary metabolites for human health are abundantly present in radish vegetable. Radish is a member of Brassicaceae family and its seed, leaf and root contain very important GSLs. The objective of this study was to determine the variation of individual and total GSL contents in leaves and roots of 44 radish (Raphanus spp.) germplasm (26 R. sativus L., 3 R. raphanistrum, and 15 R. sativus L. var. raphanistroides Makino), and compare the GSL contents between leaves and roots among three Raphanus species. Thirteen GSLs were identified, being the glucoraphasatin (GRS) and glucobrassicin (GBS) the most abundant aliphatic and indolyl GSLs in both the leaves and roots. Variation in individual and total GSL contents was found among the germplasm of three Raphanus species. The GRS content was higher in roots than that of leaves in all three Raphanus species but the GBS content was higher in leaves than roots. GRS was represented 87.0%, 92.7% and 94.7% of the total GSL in roots of R. sativus L., R. raphanistrum and R. sativus L. var. raphanistroides (Makino) germplasm, respectively. Germplasm of R. raphanistrum exhibited the highest (average, 79.5 μmol/g dw) total GSL with a ranged from 62.7 to 92.9 μmol/g dw. The germplasm IT119288, Joseonmu and IT119262 from R. sativus L., RA 504 and K046542 from R. raphanistrum, and Gyeongju-2003-32 (G2003-32) and IT302373 from R. sativus L. var. raphanistroides (Makino) had high total GSL contents and these could be good candidates for developing the functional compounds-rich varieties in radish breeding program.