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Fungi produce a wide diversity of secondary metabolites with interesting biological activities for the health, industrial, and agricultural sectors. While fungal genomes have revealed an unexpectedly high number of biosynthetic pathways that far exceeds the number of known molecules, accessing and characterizing this hidden diversity remain highly challenging. Here, we applied a combined phylogenetic dereplication and comparative genomics strategy to explore eight lichenizing fungi. The determination of the evolutionary relationships of aromatic polyketide pathways resulted in the identification of an uncharacterized biosynthetic pathway that is conserved in distant fungal lineages. The heterologous expression of the homologue from Aspergillus parvulus linked this pathway to naphthalenone compounds, which were detected in cultures when the pathway was expressed. Our unbiased and rational strategy generated evolutionary knowledge that ultimately linked biosynthetic genes to naphthalenone polyketides. Applied to many more genomes, this approach can unlock the full exploitation of the fungal kingdom for molecule discovery. IMPORTANCE Fungi have provided us with life-changing small bioactive molecules, with the best-known examples being the first broad-spectrum antibiotic penicillin, immunosuppressive cyclosporine, and cholesterol-lowering statins. Since the 1980s, exploration of chemical diversity in nature has been highly reduced. However, the genomic era has revealed that fungal genomes are concealing an unexpected and largely unexplored chemical diversity. So far, fungal genomes have been exploited to predict the production potential of bioactive compounds or to find genes that control the production of known molecules of interest. But accessing and characterizing the full fungal chemical diversity require rational and, thus, efficient strategies. Our approach is to first determine the evolutionary relationships of fungal biosynthetic pathways in order to identif