Grifola frondosa is a commercially important edible mushroom, yet the molecular mechanisms underlying fruiting body development in this species remain poorly understood. In this study, five sample types were collected from the same cultivation bag bearing mature fruiting bodies to represent different stages of a developmental continuum: spawn (S), white mycelial film (WM), gray mycelial mat (GM), fruiting body base (B), and pileus (P). Transcriptomic, biochemical, and morphological analyses were integrated to investigate the tissue-specific characteristics and potential roles of these tissues during fruiting body formation. The results revealed that S exhibited enhanced cellulase expression and activity, consistent with its proposed role in carbon supply for the developing fruiting body. WM showed upregulation of genes involved in H₂O₂ production and ATP synthesis, together with hydrophobin-related genes, consistent with active energy metabolism and a possible role in surface protection in this light-exposed layer. GM displayed distinct morphological features, including thicker hyphae with rounded tips and elevated expression of lectin, transcription-related, and signal transduction genes, consistent with its possible role as the site of primordium initiation. Both B and P specifically upregulated meiosis-related genes, suggesting their involvement in spore production. Collectively, these findings provide a comprehensive molecular resource for understanding tissue-specific genetic programs governing fruiting body formation in G. frondosa, while also highlighting candidate genes and testable hypotheses for future functional validation.