In comparison with other cruciferous vegetables, horseradish has rarely been the object of scientific research, and the knowledge about the composition, content and distribution of glucosinolates (GLS) in different organs of horseradish plants is limited. Therefore, the aim of this study was to evaluate changes in the GLS content in leaves and roots of four horseradish landraces during the growing season. The presence of 13 GLS was determined in the examined horseradish tissues, and glucoraphanin, glucoraphenin and napoleiferin were noted for the first time in the species. During the growing season, the content of individual GLS changed significantly. The rate and direction of these changes varied across the examined landraces and plant organs. In the leaves, between May and June, the content of sinigrin, the main GLS in all horseradish landraces, decreased in Bavarian (40%) and Hungarian (11%) horseradish, increased (22%) in Creamy horseradish, whereas in Danish horseradish, the difference was not significant. Despite the changes observed in the first two months, the highest content of sinigrin was noted in July in all horseradish landraces. During the growing season (August-October), the content of sinigrin fluctuated in the roots of Creamy and Danish landraces, reaching the highest level in October and September, respectively, whereas in the roots of Hungarian and Bavarian landraces, sinigrin concentrations continued to increase and peaked in October. Changes in the content of other, minor GLS during the growing season often differed from those noted in sinigrin levels.