AbstractMultiple resistance to glyphosate, sethoxydim, and paraquat was previously confirmed in two Italian ryegrass [Lolium perenneL. ssp.multiflorum(Lam.) Husnot] populations,MR1andMR2, in northern California. Preliminary greenhouse studies revealed that both populations were also resistant to imazamox and mesosulfuron, both of which are acetolactate synthase (ALS)-inhibiting herbicides. In this study, three subpopulations,MR1-A(from seed ofMR1plants that survived a 16X rate of sethoxydim),MR1-P(from seed ofMR1plants that survived a 2X rate of paraquat), andMR2(from seed ofMR2plants that survived a 16X rate of sethoxydim), were investigated to determine the resistance level to imazamox and mesosulfuron, evaluate other herbicide options for the control of these multiple resistantL. perennessp.multiflorum, and characterize the underlying ALS-inhibitor resistance mechanism(s). Based on LD50values, theMR1-A,MR1-P, andMR2subpopulations were 38-, 29-, 8-fold and 36-, 64-, and 3-fold less sensitive to imazamox and mesosulfuron, respectively, relative to the susceptible (Sus) population. OnlyMR1-P and MR2plants were cross-resistant to rimsulfuron, whereas bothMR1subpopulations were cross-resistant to imazethapyr. Pinoxaden (ACCase inhibitor [phenylpyrazoline 'DEN']) only controlledMR2andSusplants at the labeled field rate. However, all plants were effectively controlled (>99%) with the labeled field rate of glufosinate. Based on I50values,MR1-A,MR-P, andMR2plants were 712-, 1,104-, and 3-fold and 10-, 18-, and 5-fold less responsive to mesosulfuron and imazamox, respectively, than theSusplants. Sequence alignment of theALSgene of resistant plants revealed a missense single-nucleotide polymorphism resulting in a Trp-574-Leu substitution inMR1-AandMR1-Pplants, heterozygous in both, but not in theMR2plants. An additional homozygous substitution, Asp-376-Glu, was identified in theMR1-Aplants. Addition of malathion or piperonyl butoxide did not alter the efficacy of mesosulfuron onMR2plants. In addition, the presence of 2,4-D had no effect on the response of mesosulfuron on theMR2andSus. These results suggest an altered target site is the mechanism of resistance to ALS inhibitors inMR1-AandMR1-Pplants, whereas a non–target site based resistance apparatus is present in theMR2plants.