With the frequency of glyphosate-resistant waterhemp increasing throughout the Midwest, the identification of resistant populations has become important for managing this species. However, high-throughput screening for glyphosate resistance in the greenhouse is tedious and inefficient. Research was conducted to document the occurrence of glyphosate-resistant waterhemp throughout the state of Illinois, and to determine whether a molecular assay for 5-enolypyruvyl-shikimate-3-phosphate synthase (EPSPS) gene amplification can be used as an alternative means to detect resistant populations. Populations throughout the state of Illinois were collected in 2010 and screened for glyphosate resistance using a whole-plant assay in a greenhouse, and survivors were examined for EPSPS gene amplification. Of 80 populations investigated, 22 were glyphosate resistant based on the greenhouse screen, and gene amplification was identified in 20 (91%) of the resistant populations. Although there are multiple mechanisms for glyphosate resistance in waterhemp, a molecular test for EPSPS gene amplification provides a rapid alternative for identification of glyphosate resistance in most populations.