To identify seed glucosinolates in Japanese rapeseed (Brassica napus L.), a method for the separation and identification of various desulfoglucosinolates that were prepared by digestion with sulfatase from glucosinolates, was developed using liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (LC/APCI-MS). Desulfoglucosinolates were separated by a high-performance liquid chromatography (HPLC) system and were analyzed by APCL-MS. Nine desulfoglucosinolates were easily identified from the protonated molecular ions (M + N)⁺ and the group-specific fragment ions (M + H - C₆H₁₀O₅)⁺. The seed of Japanese rapeseed contained the same glucosinolates as the exotic cultivars. These results revealed that LC/APCI-MS analysis is suitable for the identification of desulfoglucosinolates. HPLC analysis showed that there were remarkable varietal differences in the total and individual glucosinolate contents among the 50 main Japanese cultivars. Among these cultivars, Norin 18 showed the lowest glucosinolate content.