Wet sieving of soil samples, followed by plating on semi-selective medium and microscopic analysis, is the most commonly used technique to quantify microsclerotia-forming Verticillium species in soil. However, the method is restricted to small samples, does not allow easy differentiation between species, and takes several weeks to complete. This study describes an alternative method to test 100-g soil samples for three Verticillium species (V. tricorpus, V. dahliae, and V. longisporum) using density flotation-based extraction of microsclerotia followed by new real-time polymerase chain reaction (PCR) assays. Primers for these real-time PCR assays were designed to the ribosomal DNA internal transcribed spacer for V. tricorpus and the β-tubulin gene for V. dahliae + V. longisporum and V. longisporum. Tests with artificially and naturally infested soils showed that the new method is reproducible and sensitive (0.1 to 0.5 microsclerotia/g soil), allows differentiation among the three species, and can be completed in one day. The results of the new method and the wet-sieving method were highly correlated for V. tricorpus (R2 = 0.78), but not for V. dahliae/V. longisporum, probably due to the loss of germinability of V. dahliae/V. longisporum microsclerotia during prolonged dry storage of the soil.