We describe a polymerase chain reaction (PCR)-based test that is specific for the pathogenic European biotype 2 (Th2) and North American biotype 4 (Th4) of Trichoderma harzianum, responsible for the green mold epidemic in the cultivated mushroom, Agaricus bisporus. A PCR primer pair was designed that targets a 444-bp arbitrary sequence in the genome of Th4. The primers also amplified the same product with Th2, but showed no reactivity with other biotypes of T. harzianum, several biocontrol Trichoderma, or with 31 other genera and species of fungi. The PCR-based test should have application in disease management programs, and in the evaluation of biocontrol Trichoderma for potential pathogenicity on mushrooms.