Grosmannia clavigera is a fungal pathogen associated with the mountain pine beetle (Dendroctonus ponderosae) which is devastating large areas of western Canada's conifer forests. This fungus also produces a dark melanin pigment that discolors pine sapwood. We have generated the draft genome of G. clavigera. However, functional characterization of genes identified in the genome sequence requires an efficient gene disruption method. In this work, we report a gene replacement strategy for G. clavigera using the Agrobacterium-mediated transformation in conjunction with linear or split-marker deletion cassettes. In addition, we used long flanking regions up to 3 kb from both sides of the targeted genes in our deletion cassettes. We assessed this gene disruption method with two genes from the melanin biosynthesis pathway that produce easily detectable white and red/brown mutant phenotypes: polyketide synthase and scytalone dehydratase. The approach yielded G. clavigera gene replacements with homologous recombination rates between 65 and 82%. For filamentous fungi, this is the first report showing that split-markers can be used with Agrobacterium-mediated transformation to generate appropriate mutants. This method can now be applied to efficiently identify genes involved in G. clavigera fungal pathogenicity and will facilitate understanding how the fungus overcomes the host defence system.