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In Europe, glyphosate-resistant weeds have so far only been reported in perennial crops. Following farmers' complaints of poor herbicide efficacy, resistance to glyphosate as well as to ACCase and ALS inhibitors was investigated in 11 populations of Lolium spp. collected from annual arable cropping systems in central Italy. Field histories highlighted that farmers had relied heavily on glyphosate, often at low rates, as well as in a non-registered crop. The research aimed at elucidating the resistance status, including multiple resistance, of Lolium spp. populations through glasshouse screenings and an outdoor dose-response experiment. Target-site resistance mechanism was also investigated for the substitutions already reported for EPSPs, ALS and ACCase genes. Three different resistant patterns were identified: glyphosate resistant only, multiple resistant to glyphosate and ACCase inhibitors and multiple resistant to glyphosate and ALS inhibitors. Amino acid substitutions were found at position 106 of the EPSPs gene, at position 1781, 2088 and 2096 of the ACCase gene and at position 197 and 574 of the ALS gene. Not all populations displayed amino acid substitutions, suggesting the presence of non-target-site-mediated resistance mechanisms. After 39 years of commercial availability of glyphosate, this is the first report of multiple resistance involving glyphosate selected in annual arable crops in Europe. Management implications and options are discussed. [ABSTRACT FROM AUTHOR]