Ophiostoma species are an economically important group of saprophytic and pathogenic fungi that grow in trees or wood. Ophiostoma like O. piceae and O. floccosum produce melanin, a pigment that stains lumber and logs. We used such species as model organisms for characterizing the molecular mechanisms in fungal melanin production. Because homologous recombination is rare in the Ophiostoma, identifying gene function in this group is challenging. We addressed this by assessing RNA interference (RNAi) as an alternative to gene replacement. For this, we built different inverted repeat transgene (IRT) constructs to down-regulate the polyketide synthase (PKS1) gene of the melanin pathway in O. piceae and O. floccosum. Transformation with IRT-PKS reduced mRNA levels for the PKS1 gene, and consequently decreased pigmentation in transformants. We showed that the PKS1 RNAi efficiency was proportional to the length of the dsRNA expressed from IRT constructs. These results indicated that RNAi is an appropriate tool for functional analysis of genes in Ophiostoma.