ABSTRACT Soybean root rot is a major disease of soybean (Glycine max [L.] Merr.) under continuous cropping, which leads to dramatic variations in the rhizosphere microflora. Soybean was sown in a different field after year zero, and continuous cropping was applied for 1 or 2 yr. The objectives were to investigate the variation in fungal populations present or inhabiting soybean roots during 3 yr of monocropping using next-generation sequencing to compare the three sets of root samples and provide a theoretical basis for the following inoculation study of the different pathogens involved in root rot disease in soybean, variation in fungal populations, and incidence of root rot. Results showed that operational taxonomic units (OTUs) of the three samples were divided into 19 phyla, 169 families, and 235 genera. Ascomycota and Basidiomycota were the dominant phyla in the continuous cropping root samples. Continuous cropping could increase the relative abundance of some fungi, namely Fusarium, Rhizoctonia, and Thelebolus, which are associated with soybean root rot for 2 and 3 yr of cropping. Continuous cropping could also increase the abundance of Gymnoascus, Chrysosporium, Ctenomyces, Aphanoascus, and Aspergillus, which are soil pathogenic fungi that can cause other plant diseases