It is known that the glycerol phosphate pathway serves as the fundamental mechanism of triglyceride biosynthesis in many organisms and plays a critical role in the synthesis of fatty acids and phospholipids, in addition to energy production. I In this study, a comprehensive analysis of the PlsC (GPAT) gene family within the Anopheles gambiae genome was conducted for the first time, and 62 distinct PlsC genes were identified from Musca domestica, Anopheles stephensi, Culex quinquefasciatus, and Drosophila melanogaster. Through phylogenetic analyses, the evolutionary relationships of PlsC genes in An. gambiae and other insect species were elucidated. Furthermore, the protein structures, chromosomal locations, three-dimensional models, and protein-protein interactions of these genes were investigated. The results indicated that although most proteins were determined to be targeted to the endoplasmic reticulum, mitochondria, cytoplasm, or cell membrane, they often lacked distinct organelle targeting signals. Additionally, current findings suggest that the PlsC gene family may play a role in adaptation to environmental stress factors and insecticide tolerance. These comprehensive findings provide significant insights into the evolutionary processes and modifications of the PlsC gene family and contribute to a better understanding of biological processes such as growth, development, and thermal resistance in vector organisms.