RNA-mediated gene silencing has been demonstrated in plants, animals, and more recently in filamentous fungi. Here, we report high frequency, RNA-mediated gene silencing in the apple scab fungus, Venturia inaequalis. The green fluorescent protein (GFP) transgene was silenced in a GFP-expressing transformant. An endogenous gene, trihydroxynaphthalene reductase (THN), involved in melanin biosynthesis, was also silenced. Silencing of these two genes resulted in obvious phenotypes in vitro. High frequency gene silencing was achieved using hairpin constructs for the GFP or the THN genes transferred by Agrobacterium (71 and 61%, respectively). THN-silenced transformants exhibited a distinctive light brown phenotype and maintained the ability to infect apple. Of significance was the simultaneous silencing of the two genes from a single chimeric, inverted repeat hairpin construct. Silencing of both genes with this construct occurred at a frequency of 51% of all the transformants. All 125 colonies silenced for the GFP gene were also silenced for THN. As THN and GFP silenced transformants have readily detectable phenotypes, the genes have utility as markers for gene silencing. Simultaneous, multiple gene silencing, utilising such marker genes, will enable the development of high through-put screening for functional genomics. This chimeric technology will be particularly valuable when linked with silenced genes that have no obvious phenotype in vitro.