The root-lesion nematode Pratylenchus penetrans is a major pathogen of potato worldwide. Yield losses may be exacerbated by interaction with the fungus Verticillium dahliae in the potato early dying disease complex. Accurate identification and quantification of P. penetrans prior to planting are essential for developing effective integrated pest control measures. However, distinction between P. penetrans and other Pratylenchus spp. based on morphology is a tedious task. A SYBR Green I-based qPCR assay was developed to discriminate, identify, and quantify P. penetrans in field soil. P. penetrans-specific qPCR primers were designed from the D2-D3 region of the 28S rDNA. The specificity of the assay was evaluated using eight isolates of P. penetrans populations and 31 isolates of other nematode species. A standard curve relating threshold cycle and log values of nematode number was generated from artificially infested soils. There was a high correlation between the P. penetrans numbers artificially added to soil or estimated from naturally infested field soils by conventional methods, and the numbers quantified using the qPCR assay. Grinding the field soil prior to DNA extraction improved P. penetrans detection from soil. The qPCR assay will not only be useful for differentiating P. penetrans from mixed populations of Pratylenchus spp., but also for efficient detection and quantification of P. penetrans from field soil. The assay requires no expertise in nematode taxonomy and morphology, and may serve as a useful diagnostic tool in research, diagnostic labs, and extension services for pest management.