Italian ryegrass [Lolium perenne ssp. multiflorum (Lam.)] Husnot]; referred as L. multiflorum throughout the manuscript hereafter) is one of the most problematic grass weeds infesting agronomic and specialty crops across the United States. In 2023–2025, inadequate control of L. multiflorum populations (NY_R1, NY_R2, and NY_R3) with glyphosate was reported in Livingston, Ontario, and Genesee Counties, New York (NY). This research aimed to (1) confirm and quantify glyphosate resistance in these suspected glyphosate-resistant (GR) populations, (2) evaluate the efficacy of alternative postemergence herbicides, and (3) determine whether EPSPS gene amplification confers glyphosate resistance. A known glyphosate-susceptible (GS) population (AR_S) from Arkansas was included for comparison. Glyphosate dose–response assays indicated that NY_R1, NY_R2, and NY_R3 populations were 13-, 4-, and 5-fold resistant, respectively, relative to the AR_S population. Alternative postemergence herbicides, including clethodim, glufosinate, paraquat, and pinoxaden, provided 96 to 97% control and reduced shoot dry weight by 91 to 97% at 21 days after treatment (DAT). In contrast, nicosulfuron provided reduced control (63 to 74%) and limited biomass reduction (51 to 56%), suggesting possible resistance to acetolactate synthase (ALS)-inhibiting herbicides in three tested populations. Quantitative PCR analysis revealed that NY_R1 and NY_R3 had approximately 30-fold higher EPSPS gene copy numbers than AR_S, indicating gene amplification as a mechanism of glyphosate resistance. This study confirms the first case of GR L. multiflorum associated with EPSPS gene amplification in NY, underscoring the need for integrated, diversified weed management strategies to mitigate its further spread.