Technical Abstract: Resistance to glyphosate in weed species is a major challenge for the sustainability of glyphosate use in crop and non-crop systems, and especially in glyphosate-resistant crops. A glyphosate-resistant Italian ryegrass population has been identified in Arkansas. This research was conducted to elucidate its resistance mechanism. We investigated resistant and susceptible plants from a population in Desha County, Arkansas (Des03).The amounts of glyphosate that reduced growth of resistant plants 50% (GR50) were 7 to 13 times greater than those of susceptible plants from the same population. The EPSPS gene did not contain any point mutation that has previously been associated with resistance to glyphosate, nor were there any other mutations on the EPSPS gene unique to the Des03 resistant plants. The resistant plants had 6-fold higher basal EPSPS enzyme activities than the susceptible plants, but their I50 values in response to glyphosate were similar. The resistant plants contained up to 25 more copies of EPSPS gene than the susceptible plants. The level of resistance to glyphosate correlated with increases in EPSPS enzyme activity and EPSPS copy number. Increased EPSPS enzyme activity and EPSPS gene amplification confer resistance to glyphosate in Des03 population. This is the first report of EPSPS gene amplification in glyphosate-resistant Italian ryegrass. Other resistance mechanism(s) may also be involved.