Glyphosate is the world's most widely used herbicide. It is nonselective and has been used to control a broad range of weed species for the past 20 yr, without the appearance of resistant weed biotypes. However, a biotype ofLolium rigidumfrom a field in Northern Victoria, Australia, in which glyphosate had been used for the past 15 yr, failed to be controlled by label recommended rates. Based on LD₅₀values from pot dose-response experiments, this biotype exhibited resistance to glyphosate and was nearly 10-fold more resistant compared to the susceptible biotypes tested. The biotype was resistant to three different salts of glyphosate. The biotype was also nearly threefold more resistant to diclofop-methyl but was susceptible to other commonly used selective and broad-spectrum herbicides. Between the two-leaf and tillering stages of development, a susceptible biotype exhibited a small but significant decrease in tolerance to glyphosate, whereas tolerance of the resistant biotype remained unchanged with age. The resistant phenotype was verified in experiments in which seed was germinated in the presence of glyphosate. Observations on shoot and root growth of seedlings in these experiments suggested that the resistance mechanism might be associated more with the shoot than with the root.