Technical Abstract: Verticillium dahliae is responsible for Verticillium wilt on a wide range of hosts including strawberry, on which low inoculum densities can cause significant crop loss. Determination of inoculum density is currently done by soil plating, but this can take 6-8 weeks to complete and delay the grower’s ability to making planting decisions. To provide a faster means for estimating pathogen populations in the soil a multiplexed TaqMan real time PCR assay based on the rDNA intergenic spacer (IGS) was developed for V. dahliae. The assay was highly specific for V. dahliae and included an internal control for evaluation of inhibition of amplification efficiency due to the presence of PCR inhibitors in soil extracted DNA. An excellent correlation between real time PCR results and inoculum densities determined by soil plating in a range of field soils was observed in regression analysis (R2 = 0.96) with pathogen densities as low as 1-2 MS/g soil accurately detected. A TaqMan V. tricorpus assay was developed to help differentiate colonies of this species on soil plates. Variation in copy number of the rDNA IGS was also evaluated among isolates by SYBR Green real time PCR amplification of the V. dahliae specific amplicon compared to amplification of several single copy genes and was estimated to vary from approximately 24 to 73 copies per haploid genome, which translated into possible differences in results among isolates averaging 1.8 Ct. Analysis of the variation in results of V. dahliae quantification among extractions of the same soil sample indicated assaying 4 replicate DNA extractions for each field sample would provide accurate results.