Chronic stress poses a significant challenge to aquaculture production, and cortisol and glucose have traditionally served as stress markers. However, recent doubts have emerged about the reliability of scale cortisol as a chronic stress determinant due to its involvement in calcium homeostasis. While cortisol influences gene expression in the stress response cascade, few studies explore its impact on metabolic pathways in cultured Nile tilapia. This study delves into the relationship between cortisol signatures of chronic stress in Nile tilapia and the regulation of the Mitogen Activated Protein Kinase (MAPK) signalling pathway, crucial for cell processes. Juvenile Nile tilapia were subjected to different ammonia concentrations and stocking densities for 70 days, with assessments of growth performance, stress levels, RNA sequencing, and differential gene expression. The results revealed a positive correlation between stressors and blood glucose, plasma cortisol, and scale cortisol concentrations. Fish in water with 0.8 mg/L ammonia exhibited elevated plasma glucose and cortisol, while those in 1.2 mg/L showed increased scale cortisol. Higher stocking densities also correlated with elevated stress markers. Notably, cortisol levels rose with ammonia concentration and stocking density, negatively impacting growth performance. The MAPK signalling pathway, linked to cell processes, showed significant downregulation with increasing ammonia concentrations, suggesting its sensitivity to stress. Taken together, six genes in this pathway were significantly enriched following ammonia treatment (Padj less than or equal to 0.05) i.e. Dual Specific Protein Phosphatase 1 (DUSP1), Nuclear Hormone Receptor 38 (NHR38), Heat Shock Protein 1 (HSP 72 KDa), Myelocytomatosis oncogene homologue (Mych), Growth arrest and DNA damage inducible alpha a (GADD 45aa) and Mitogen Activated Protein Kinase 4 (MAP2K4). This study contributes to unravelling the intricate relationship between chronic stress, cortisol, and the MAPK pathway in aquaculture, providing insights crucial for optimizing fish welfare and production.