ABSTRACT Rusted root (also known as rusty root) of ginseng (Panax quinquefolius) was first described over 70 years ago, but the causal agent has not been clearly established. The disease is characterized by slightly raised reddish-brown to black root lesions of varying size. The lesions, regardless of size, remain superficial; however, peridermal tissue is ruptured and sloughed off, giving the root a scabbed appearance. Culture-independent techniques were used to demonstrate that a fungal internal transcribed spacer (ITS) region DNA fragment was strongly associated with diseased but not healthy root tissue. The fragment ( approximately 650 bp in length) was cloned. Restriction enzyme digests of cloned DNA indicated that the 650-bp fragment represented a single taxon. BLAST analysis following sequencing of the fragment found that the nearest matches in GenBank were anamorphic genera associated with discomycetes, in particular Rhexocercosporidium spp. This putative identification was supported further by isolating fungi from diseased tissue using a semiselective agar medium. With this procedure, a Rhexocercosporidium-like fungus was isolated; DNA extracted from fungal cultures and amplified using ITS oligonucleotide primers was found to be identical to similarly amplified DNA from the 650-bp bands. However, the isolates were distinct, with respect to growth rate on agar media and ITS sequence, from Rhexocercosporidium carotae, the only described species in this genus. The ability to reproduce symptoms on ginseng roots was confirmed in pathogenicity tests. Oligonucleotide primers based on ITS sequences were designed to amplify DNA of Rhexocercosporidium spp. Polymerase chain reaction assays on DNA extracted from naturally infected root tissue showed that the fungus was present in nearly all symptomatic roots but was infrequent in healthy-appearing roots. The most probable cause of rusted root of ginseng is a previously undescribed species of Rhexocercosporidium.