Abstract: To provide a scientific basis for control of Rheum palmatum root rot, potential pathogens were isolated from rotted roots and identified before use in root segment inoculation pathogenicity tests. Eight species of fungi were isolated from rotted roots of biennial R. palmatum cultivated in Lixian county of Gansu. The chief pathogens were Trichothecium roseum, Fusarium oxysporum, and Rhizotonia solani. Their isolation frequencies varied with the pathogens and plant growth stage. T. roseum and R. solani could be isolated from different growth stages with frequencies of 41.18%-55.56% and 3.03%-44.44%, respectively. However, F. oxysporum could only be isolated from plants in the late growth stages, with an isolation frequency of 9.09%. The incidence of root rot after inoculation of pathogens into wounded radicles was significantly higher than that into non-wounded radicles. The incubation period of F. oxysporum was short with large disease spots indicating a strong pathogenic ability, while the incubation period of T. roseum and R. solani were long with small spots indicating a weak pathogenic ability. All the above shows that wounds are helpful for root rot pathogens to infect the plants. It suggests that transplanting and cultivation should be done carefully to maintain the plant integrity and prevent wounding. In addition, root segment inoculation is easy to do and may be extensively used in plant root disease research for its short inoculation time, high re-isolation frequency, and reliable result.