Serpula wood-decay fungi occupy a diverse range of natural and man-made ecological niches. Serpula himantioides is a forest-floor generalist with global coverage and strong antagonistic ability, while closely related species Serpula lacrymans contains specialist sister strains with widely differing ecologies. Serpula lacrymans var. shastensis is a forest-floor specialist in terms of resource preference and geographic coverage, while Serpula lacrymans var. lacrymans has successfully invaded the built environment and occupies a building-timber niche. To increase understanding of the cellular machinery required for niche adaptation, a detailed study of the P450 complement of these three strains was undertaken. Cytochrome P450 monooxygenases are present in all fungi and typically seen in high numbers in wood decay species, with putative roles in breakdown of plant extractives and lignocellulose metabolism. Investigating the genomes of these related yet ecologically diverse fungi revealed a high level of concordance in P450 complement, but with key differences in P450 family representation and expression during growth on wood, suggesting P450 proteins may play a role in niche adaptation. Gene expansion of certain key P450 families was noted, further supporting an important role for these proteins during wood decay. The generalist species S. himantioides was found to have the most P450 genes with the greatest family diversity and the highest number of P450 protein families expressed during wood decay.