[Background] The increasingly serious root rot has posed a threat to the production of naked barley (Hordeum vulgare L. var. nudum Hook. f.) and hampered the prevention and control of the disease and the development of the naked barley industry in Qinghai province. However, the root rot of naked barley has been rarely studied and the pathogens are unclear. [Objective] This paper aims to clarify incidence and pathogens of the root rot in naked barley and the pathogencity of the pathogens, which is expected to lay a theoretical basis for the prevention and control of the disease. [Methods] The conventional method of tissue isolation was adopted to isolate pathogens of root rot in naked barley. The pathogens were detected by both morphological identification and molecular identification, and their pathogenicity was determined with a beaker of water-agar. [Results] A total of 4 stains with strong and significantly different pathogenicity were isolated, which were identified as Clonostachys rosea. As verified by Koch's postulates, they were new pathogens of root rot of naked barley, and the induced root rot was found for the first time at home and abroad.[Conclusion]C. rosea can cause root rot in naked barley with strong pathogenicity.