With the full genome sequence of Agaricus bisporus available, it was possible to investigate the genes involved in the melanin biosynthesis pathway of button mushrooms. Based on different BLAST and alignments, genes were identified in the genome which are postulated to be involved in this pathway. Seven housekeeping genes were tested of which 18S rRNA was the only housekeeping gene that was stably expressed in various tissues of different developmental stages. Gene expression was determined for most gene homologs (26 genes) involved in the melanin pathway. Of the analysed genes, those encoding polyphenol oxidase (PPO), the PPO co-factor L-chain (unique for A. bisporus), and a putative transcription factor (photoregulator B) were among the highest expressed in skin tissue. An in depth look was taken at the clustering of several PPO genes and the PPO co-factor gene on chromosome 5, which showed that almost 25% of the protein encoding genes in this cluster have a conserved NACHT and WD40 domain or a P-loop nucleoside triphosphate hydrolase. This article will be the start for an in depth study of the melanin pathway and its role in quality losses of this economically important product.