Wooden Breast (WB) abnormality represents one of the major challenges that the poultry industry has faced in the last 10 years. Despite the enormous progress in understanding the mechanisms underlying WB, the precise initial causes remain to be clarified. In this scenario, the present research is intended to characterize the gene expression profiles of broiler Pectoralis major muscles affected by WB, comparing them to the unaffected counterpart, to provide new insights into the biological mechanisms underlying this defect and potentially identifying novel genes likely involved in its occurrence. To this purpose, data obtained in a previous study through the RNA-sequencing technology have been used to identify differentially expressed genes (DEGs) between 6 affected and 5 unaffected broilers' breast muscles, by using the newest reference genome assembly for Gallus gallus (GRCg7b). Also, to deeply investigate molecular and biological pathways involved in the WB progression, pathways analyses have been performed. The results achieved through the differential gene expression analysis mainly evidenced the downregulation of glycogen metabolic processes, gluconeogenesis, and tricarboxylic acid cycle in WB muscles, thus corroborating the evidence of a dysregulated energy metabolism characterizing breasts affected by this abnormality. Also, genes related to hypertrophic muscle growth have been identified as differentially expressed (e.g., WFIKKN1). Together with that, a downregulation of genes involved in mitochondrial biogenesis and functionality has been detected. Among them, PPARGC1A and PPARGC1B chicken genes are particularly noteworthy. These genes not only have essential roles in regulating mitochondrial biogenesis but also play pivotal roles in maintaining glucose and energy homeostasis. In view of that, their downregulation in WB-affected muscle may be considered as potentially related to both the mitochondrial dysfunction and altered glucose metabolism in WB muscles, and their key involvement in the molecular alterations characterizing this muscular abnormality might be hypothesized.