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Abstract: Sclerotinia stem rot, caused by Sclerotinia sclerotiorum, is a major disease of canola (Brassica napus) commonly managed by the routine application of fungicides. Petal infestation is an important stage of the disease cycle and has been the focus of previously developed Sclerotinia stem rot risk assessment methods. Quantitative PCR (qPCR) analysis can provide a more accurate and rapid assessment of petal infestation level. A hydrolysis probe-based S. sclerotiorum-specific qPCR assay was developed that could detect as little as 8.0 × 10-4 ng of S. sclerotiorum DNA with a high degree of specificity. Petal infestation level, as determined with this assay at full bloom (40-50% flower), accounted for 60-92% of the variation in Sclerotinia stem rot incidence under field conditions in western Canada. The strength of the relationship, however, varied from year-to-year and over the flowering period. Petal infestation level was influenced by petal age and by the time of day when samples were collected. A diurnal fluctuation in infestation levels was found in young, but not old petals, with significantly higher infestation in afternoon- versus morning-collected samples. Sclerotinia stem rot development also was influenced by environmental conditions, with relative humidity playing a more significant role in disease development than temperature. Inclusion of relative humidity conditions in a forecasting system with petal infestation levels may improve the reliability and accuracy of Sclerotinia stem rot risk assessments. In addition to its potential utility as a risk assessment tool, the S. sclerotiorum-specific qPCR assay may also prove useful in studies of the epidemiology of Sclerotinia stem rot.