Glufosinate is an important and widely used non-selective herbicide active on a wide range of plant species. Resistance evolution to glufosinate in weedy plant species (including the global weed Eleusine indica) is underway. Here, we uncover the molecular basis of target-site glufosinate resistance in Eleusine indica. Full-length E. indica glutamine synthetase (GS) iso-genes (EiGS1-1, 1-2, and 1-3 and EiGS2) were cloned, and expression of EiGS1-1 and EiGS1-2 was higher than EiGS2. A novel point mutation resulting in a Ser-59-Gly substitution in EiGS1-1 was identified in glufosinate-resistant (R) plants. Rice calli and seedlings transformed with the mutant EiGS1-1 gene were resistant to glufosinate. Purified yeast recombinant mutant EiGS1-1 was more tolerant to glufosinate than the wild type (WT) variant. These transgenic results correlate with a more glufosinate-R GS in the crude tissue extract of R versus susceptible E. indica plants. Structural modelling of the mutant EiGS1-1 revealed that the Ser-59 is not directly involved in glufosinate binding but in contact with some important binding residues (e.g. Glu-297) and especially with Asp-56 that forms an intratoroidal contact interface. Importantly, the same Ser-59-Gly mutation was also found in geographically isolated glufosinate-R populations from Malaysia and China, suggesting parallel evolution of this resistance mutation.