Detection and quantification of airborne ascospores as a component of the Sclerotinia rot of carrot (SRC) forecast model is currently accomplished using the blue plate test (BPT), which uses Sclerotinia semiselective medium (SSM). A quantitative polymerase chain reaction (qPCR) assay was developed to reduce the time to specifically quantify ascospores of Sclerotinia sclerotiorum from air samples collected using a Burkard Multi-Vial Cyclone Sampler. The qPCR assay was highly sensitive and detected DNA from 0.5 to 5 × 104 ascospores within a linear range (R2 = 0.99). The qPCR assay was used to quantify ascospores of S. sclerotiorum in air samples collected over three growing seasons. Initial SRC disease was observed 8 and 34 days following detection of 9.5 and 2 ascospores m–3 of air, respectively. Results from air samples collected using an Andersen N6 Sampler and the qPCR assay were compared with the BPT. Ascospore counts from a Burkard Sampler coupled with the qPCR assay and the BPT followed similar trends. In general, fewer ascospores were detected and bioaerosol sampling efficiency was low using an Anderson Sampler. Three days were required to confirm the number of ascospores using SSM in the BPT and with an Andersen Sampler, whereas results from a Burkard Sampler coupled with the qPCR assay can provide results within 5 h of air sampling. The choice of method will depend on the available resources.