Improvement of cultivar resistance is the key strategy to control the host-specialized pathogen Verticillium longisporum in oilseed rape (OSR). A special feature of this pathogen is its systemic, non-homogenous and delayed colonization of the plant xylem resulting in an extended symptomless period of latency. As a result, severity of infection in the field is difficult to score as it becomes apparent only at crop maturity stages when it may be confused with natural senescence. Assessment of Verticillium disease severity in OSR by visual scoring of microsclerotia on harvested stubbles unsatisfactorily reflects genotypic resistance as it is strongly affected by the ripening stage of the plant. To overcome these limitations, we developed a qPCR method, which unambiguously differentiates levels of quantitative resistance to V. longisporum in OSR genotypes under field conditions. The specificity and sensitivity of two primer pairs targeting ITS or tubulin loci in the V. longisporum genome were tested. While tubulin primers showed a high specificity to V. longisporum isolates, ITS primers exhibited a significantly higher sensitivity in detecting fungal DNA in stem tissue (limit of quantification =0.56 fg DNA) of field-grown pre-symptomatic plants. The best discrimination of resistant and susceptible OSR cultivars based on fungal DNA analysis in stem tissue was achieved at growth stage 80, at the transition of fungal vascular growth in viable plants to saprotrophic colonization of senescent stem tissues. Field screening data obtained with qPCR at growth stage 80 confirmed results from greenhouse testing thus corroborating the relevance and reliability of seedling assays for determining cultivar responses to V. longisporum in the field, as a useful tool for breeders in first selection of elite OSR genotypes with improved resistance to Verticillium.