Brown stem rot of soybean, caused by the soilborne fungus Phialophora gregata, is a common and widespread disease of soybean (Glycine max) in the midwestern United States. This pathogen is challenging to study due to a long latent period and slow growth. A TaqMan probe-based quantitative, real-time polymerase chain reaction (qPCR) assay was developed for sensitive and specific detection and quantification of genotypes A and B of P. gregata in plant and soil samples. It is sensitive with detection limits of 50 fg of pure genomic DNA, 100 copies of the target DNA sequence, and approximately 400 conidia. The qPCR assay is approximately 1,000 times more sensitive in detecting DNA and conidia of P. gregata, and is more rapid and less sensitive to PCR inhibitors from soybean stems than a standard PCR (sPCR) assay. Using this single-step qPCR assay, low levels of infection were detected in soybean stems at least 1 to 2 weeks prior to symptom development and before P. gregata was detected with sPCR. This assay also was used to detect the pathogen in field-grown plants and in naturally infested field soils. This new qPCR assay is a powerful tool for rapid, specific, and sensitive detection, diagnosis, and quantification of P. gregata in plants and soil, and for advancing studies of the ecology of P. gregata and its interactions with host plants.