Different Brazilian sugarcane genotypes have different rates of Biological Nitrogen Fixation (BNF) and higher BNF correlates with their ability to grow with low nitrogen input. As a first step to investigate the role of sugarcane nitrogen metabolism in the good performance of Brazilian genotypes to grow in nitrogen poor soil, we identified and characterized members of glutamine synthetase (GS) family in sugarcane. Five genes were identified, encoding for cytosolic (scGS1.a, scGS1.b and scGS1.c), plastidic (scGS2) and dodecameric (scGSI) GS. These genes are differentially expressed in sugarcane organs and scGS1.a is the most expressed one. scGS1.b expression increases during shoot development, reaching high levels in mature leaves of SP70-1143 genotype (high BNF) and moderate levels in Chunee genotype (low BNF). Treatment with ammonia increased the expression of scGS1.b only in SP70-1143. In addition, withdrawal of ammonia enhanced expression of scGS1.a and scGS1.b only in SP70-1143. The data suggests that scGS1.a and scGS1.b can be important for nitrogen assimilation in sugarcane, including not only the nitrogen provided by BNF, but also supplied by soil and by remobilization. Their differential expression in SP70-1143 and Chunee genotypes could explain, at least to some extent, variations in the performance of these genotypes to grow with low nitrogen input.