Abstract Glutamine synthetase (GS) plays a fundamental role in nitrogen metabolism in higher plants. Three BnGS genes have first been isolated: one gene encoding plastid GS (BnGS2) and two encoding cytosolic GS (BnGS1-1 and BnGS1-2) in ramie. Based on a sequence analysis and phylogenetic study, three BnGS sequences were classified into three distinct sub-families. The phylogenetic analysis showed that BnGS2 and BnGS1-2 were closely related to those of legumes, alfalfa (Medicago sativa), soybean (Glycine max) and bean (Phaseolus vulgaris). The BnGS gene expression patterns revealed that each gene exhibited similar organ specificity, but distinct transcript intensity during different vegetative processes. The relatively abundant expression of BnGS1-1 and BnGS2 at specific organs during different vegetative processes indicates that they have critical roles in nitrogen uptake and assimilation relating to forage and growth characteristics. The BnGS1-2 mRNA levels were remarkably upregulated in the phloem, xylem and stems during the fiber development stage, suggesting a correlation with fiber development. Therefore, the non-overlapping transcript intensity of BnGS genes in different tissues regulates ramie growth and development during different vegetative processes.