Two cotton research institute (CRI) near-isogenic lines, CRI-12 glanded and CRI-12 glandless, were used to pinpoint potential genes and metabolic pathways linked to gossypol biosynthesis through transcriptome sequencing.  We discovered more than 235 million clean reads and 1,184 differentially expressed genes (DEGs).  Consecutively, we conducted a weighted gene co-expression network analysis and found a strong correlation between white and yellow modules containing GhTPS (GH_D09G0090) and GhCYP (GH_D05G2016) hub genes with the gossypol content.  Importance of the GhTPS and GhCYP genes was demonstrated using RT-qPCR, virus-induced gene silencing (VIGS), and target metabolite analysis.  Silencing these genes resulted in fewer glands on both leaves and stems two weeks after the infection compared to the wild type.  In addition, 152 metabolites were identified through targeted metabolite profiling.  Differential metabolite screening revealed 12 and 18 significantly different metabolites in TRV:GhTPS and TRV:GhCYP plants vs. the control group, respectively, showing a reduction in the accumulation of metabolites compared to the control.  Content of hemigossypol, the final product of gossypol biosynthesis, was also reduced, as revealed by target metabolite analysis, suggesting the role of these genes in the gossypol biosynthetic pathway.  Furthermore, a highly significant difference in gossypol content between the glanded and glandless lines was recorded.  Findings of this study reveal a strong link between the gossypol content and GhTPS and GhCYP hub genes, suggesting their role in the gossypol biosynthetic pathway to reduce the accumulation of hemigossypol, which may offer new comprehension into the regulatory checkpoints of the gossypol biosynthesis pathway in cotton.