This study was conducted to investigate the etiology of internal discoloration of horseradish roots. Several species of Fusarium and Verticillium were isolated from internally discolored horseradish roots collected from commercial fields in Illinois and research plots in Wisconsin during 2008 and 2009. Eleven isolates of Fusarium, identified as Fusarium oxysporum based on morphological features, were characterized by DNA sequencing of the nuclear translation elongation factor 1α (EF-1α) and mitochondrial small-subunit ribosomal DNA (mtSSU rDNA). Maximum parsimony analyses of DNA sequences from these two regions and the combined data set revealed that six isolates were clearly separated into a common clade that contained F. commune, with the remaining five isolates being grouped into a common clade with F. oxysporum. Based on the DNA sequence data, we considered the six isolates grouped into a common clade with F. commune to be F. commune. Pathogenicity tests of F. commune and F. oxysporum were conducted on two horseradish cultivars, ‘1573’ and ‘Big Top Western’, in a greenhouse. Root segments were inoculated by dipping them in a conidial suspension and then growing them in pots in a greenhouse for 4 months. For plants inoculated with F. commune, internal root discoloration and root rot developed 1 month after inoculation and almost all roots of the plant were completely rotten 4 months after inoculation. Inoculation of the plants with F. oxysporum resulted in only internal root discoloration but not root rot symptoms. This is the first report of F. commune causing internal discoloration and root rot of horseradish.