Root rot is an important disease hampering the sustainable cultivation of Panax notoginseng. Culture-dependent and independent techniques were used to elucidate the dominant fungal pathogen of rusty root rot of P. notoginseng. Based on Illumina sequencing profiles for fungi using ITS primers, five phyla-namely Ascomycota, Basidiomycota, Glomeromycota, Zygomycota, and Chytridiomycota-were identified, and the analyses showed that the Ascomycota was the dominant phylum (∼50 to 97%), especially in the symptomatic samples. Out of 226 total genera identified, seven genera had over 1% average abundance, including Ilyonectria, Fusarium, Tetracladium, Cladosporium, Rhizophagus, Alternaria, and Perisporiopsis. However, only Ilyonectria was the predominant genera in the symptomatic samples (∼76 to 80%), while the others, including Fusarium, had higher abundances in asymptomatic samples. Based on in vitro and in vivo pathogenicity, the isolate G3B was demonstrated to be the pathogen causing rusty root rot of P. notoginseng, and it was identified as Ilyonectria mors-panacis. Based on primers F2-R2 targeting the His3 gene of Ilyonectria, real-time quantitative PCR (qPCR) was performed as an additional proof confirming that I. mors-panacis was the dominant pathogen in the symptomatic samples during the years of the study (2014-2015).