A pair of polymerase chain reaction (PCR) primers was developed based upon the sequence of a cloned random amplified polymorphic DNA (RAPD) fragment of Alternaria radicina, and a PCR-based seed assay was developed for the detection of A. radicina from infested carrot seed. The seed assay involved a 5-day incubation step, in which seed was maintained under high humidity conditions in order to increase fungal biomass. Seed was then incubated with lysis buffer, extracted with phenol-chloroform, and DNA was recovered using a silica matrix. PCR amplification of the target A. radicina DNA sequence was enhanced by the addition of skim milk to the PCR reaction mixture. With this PCR-based seed assay, A. radicina was detected from carrot seed lots with natural infestation rates as low as 0.3%. In seed lots prepared by mixing known amounts of A. radicina-infested seed with noninfested seed, this assay allowed for the detection of the pathogen from lots with infestation rates as low as 0.1%.