The quantification of the soilborne pathogen Fusarium virguliforme inoculum in soil is important for epidemiological studies of soybean sudden death syndrome (SDS). Classical dilution plating methods to determine inoculum density in soil have yielded inconsistent results due to slow growth, variable colony morphology of the pathogen, and the presence of other fungi with similar phenotype. A TaqMan real-time polymerase chain reaction assay was developed based on sequences of the FvTox1 gene of F. virguliforme. The gene differed by four single-nucleotide proteins from the other SDS-causing species. Assay specificity was tested on 48 fungal isolates that varied in taxonomic relatedness. Assay sensitivity was appraised on 10-fold serial dilutions of genomic DNA, conidia suspensions, and soil spiked with conidia. Applicability of the assay was evaluated on field and greenhouse soil samples, and on roots of symptomatic plants. The assay detected only DNA sequences specific to F. virguliforme. The detection limit of the assay was 5 pg/μl, 1,000 conidia/ml, and 1,000 conidia/g soil for genomic DNA, conidial suspensions, and soil with conidia, respectively. The assay was specific to F. virguliforme and was used successfully to quantify inoculum density in soil and soybean roots. The assay can be used as a diagnostic tool for rapid screens of field and greenhouse soil, and for symptomatic and asymptomatic plants.