Summary Glutamine synthetase (GS; E.C.6.3.1.2) is a key enzyme in higher plants with two isozymes, cytosolic GS1 and plastidic GS2, and involves in the assimilation and recycling of NH4+ ions and maintenance of complex traits such as crop nitrogen‐use efficiency and yield. Our present understanding of crop nitrogen‐use efficiency and its correlation with the functional role of the GS family genes is inadequate, which delays harnessing the benefit of this key enzyme in crop improvement. In this report, we performed a comprehensive investigation on the phylogenetic relationship, structural properties, complex multilevel gene regulation, and expression patterns of the GS genes to enrich present understanding about the enzyme. Our Gene Ontology and protein–protein interactions analysis revealed the functional aspects of GS isozymes in stress mitigation, aging, nucleotide biosynthesis/transport, DNA repair and response to metals. The insight gained here contributes to the future research strategies in developing climate‐smart crops for global sustainability. Significance Statement Glutamine synthetase (GS) is a key enzyme involved in crop nitrogen‐use efficiency and is a determining factor for yield. Our present understanding of crop nitrogen‐use efficiency and its correlation with the functional role of GS family genes is inadequate, which delays harnessing the benefit of this key enzyme. Present report explores the extent of structural and functional properties of GS isozymes through bioinformatics approaches. In conclusion, this study summarize the present understanding as well as future perspectives of research and manipulation of GS genes/proteins.