This study established a quick and accurate method to detect petal infection of oilseed rape (Brassica napus) by Sclerotinia sclerotiorum using a nested-PCR technique. DNA samples were extracted from each petal using a microwave method, followed by two rounds of PCR amplification. The first-round PCR amplification was performed using the universal fungal primer pair ITS4/ITS5, and the second-round amplification with a specific primer pair XJJ21/XJJ222, which was designed using the single-nucleotide polymorphisms among nuclear rDNA ITS sequences of Sclerotinia spp., Botrytis spp. and other selected fungi. The established technique is rapid and inexpensive, and has a high degree of specificity and sensitivity. This assay can distinguish Sclerotinia spp. from other fungi, including Botrytis cinerea, a closely related and frequent cohabitant on oilseed rape petals, and can detect 50 fg genomic DNA, five ascospores of S. sclerotiorumin vitro or 50 ascospores of S. sclerotiorum on one petal in approximately 6 h, even in the presence of a high background of oilseed rape DNA. This technique was successfully applied in detecting natural petal infections.