Pearl millet (Pennisetum glaucum) is an important cereal crop in the arid and semiarid regions of Sub-Saharan Africa and South Asia, growing in high temperatures and irregular rainfall. It can tolerate harsh conditions and grow in low-fertility soil sets it apart from other crops. Pearl millet has been known for its high nutrient composition, which makes it a decent supply of energy for both food and fodder. Despite its value and contribution, pearl millet does not receive sufficient attention due to some anti-nutritional elements such as phytic acid, polyphenols, and tannins. Also, the rapid development of rancidity after the milling of pearl millet grains is another reason for the underutilization of pearl millet. Higher amounts of C-glycosyl flavones, including vitexin, orientin, glycosyl vitexin, and glycosyl orientin, contribute to off-flavor issues during storage and are also responsible for health problems. In this study, a genome-wide search identified 191 UGT genes in pearl millet, focusing on gene characteristics, subcellular localization, phylogenetic relationships, and protein structure. This study also aims to investigate the C-glycosyl flavone content variations among diverse varieties by HPLC. The production of C-GFs depends on the activity of the key enzyme C-glycosyltransferase (CGT) that belongs to the family 1 glycosyltransferase. Furthermore, the identification, characterization, and heterologous expression analysis of a gene coding for a C-glycosyltransferase, a key enzyme involved in C-glycosyl flavones synthesis, was done. Also, the expression analysis of this gene in different parts of the pearl millet plant was done in the present work. The results showed that a higher C-GF content was associated with a darker color and off odor of the flour. This study contributes to a comprehensive understanding of the biosynthetic pathway of C-GFs in pearl millet and provides potential interventions to improve the crop's nutritional content, ensuring its role in sustainable and resilient food systems.