Annual bluegrass (Poa annua L.) populations in turfgrass have evolved resistance to several herbicides in the United States (US), but there has been no confirmed resistance from an agricultural field. Recently, glyphosate failed to control a P. annua population found in a field in a soybean [(Glycine max (L.) Merr.] and rice (Oryza sativa L.) rotation in Poinsett County, Arkansas. The present study focused on determining the sensitivity of a putatively-resistant accession (R1) to glyphosate compared with two susceptible accessions (S1 and S2). The experiments included a dose-response study, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene copy number and expression analysis, and assessment of mutations in EPSPS. Based on the dose-response analysis, R1 required 1,038 g ae ha-1 of glyphosate to cause 50% biomass reduction, whereas S1 and S2 only required 148.2 g and 145.5 g ae ha-1, respectively. The resistant index (RI) was approximately 7-fold relative to the susceptible accessions. Real-time polymerase chain reaction data revealed at least a 15-fold increase in the EPSPS copy number in R1, along with a higher gene expression. No mutations in EPSPS were found. Gene duplication was identified as the main mechanism conferring resistance in R1. The research presented here reports the first incidence of glyphosate resistance in P. annua from an agronomic field crop situation in the US.