Chronic stress is a serious threat to aquaculture as it lowers fish growth performance and compromises fish welfare. The exact mechanism by which growth is retarded is, however, not clearly understood. This study sought to elucidate the gene expression profiles associated with chronic stress in cultured Nile tilapia (Oreochromis niloticus) reared for 70 days at different ammonia concentrations and stocking densities. Fish in the treatment groups showed negative growth, while the controls showed positive allometric growth. The specific condition factor (Kn) ranged from 1.17 for the controls to 0.93 for the ammonia and 0.91 for the stocking density treatments. RNA was extracted from muscle tissue using TRIzol followed by library construction and Illumina sequencing. Comparative transcriptome analysis revealed 209 differentially expressed genes (DEGs) (156 up- and 53 down-regulated) in the ammonia and 252 DEGs (175 up- and 77 down-regulated) in the stocking density treatment. In both treatments, 24 and 17 common DEGs were up- and down-regulated, respectively. DEGs were significantly enriched in six pathways associated with muscle activity, energy mobilization and immunity. The heightened muscular activity consumes energy which would otherwise have been utilized for growth. These results bring to fore the molecular mechanisms underlying chronic stress’ suppression of growth in cultured Nile tilapia.