Within the family of Brassicaceae, an important source of glucosinolates (GLSs) is represented by horseradish ( Armoracia rusticana P. Gaertner, B. Meyer & Scherbius), cultivated for its roots, which are grated fresh or processed into a sauce and used as a condiment. The characteristic pungent flavor of the root depends on the abundance of the bioactive GLS molecules. In crude plant extracts (sprouts and roots) of an accession of horseradish largely diffused in the Basilicata region (southern Italy), which develops many sprouts and produces white, fiery, and sharp-flavored marketable roots, we characterized the GLS profile by LC-ESI-LTQ-FTICR-MS and IRMPD. In sprouts and roots we identified 16 and 11 GLSs, respectively. We confirmed the presence of sinigrin, 4-hydroxyglucobrassicin, glucobrassicin, gluconasturtin, and 4-methoxyglucobrassicin and identified glucoiberin, gluconapin, glucocochlearin, glucoconringianin, glucosativin, glucoibarin, 5-hydroxyglucobrassicin, glucocapparilinearisin or glucobrassicanapin, glucotropaeolin, and glucoarabishirsutain, not previously characterized in horseradish. Of particular note was the presence of the putative 2-methylsulfonyl-oxo-ethyl-GLS.