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<i>Lolium</i> spp. are troublesome weeds mainly found in winter cereal crops worldwide, including Europe. In recent years resistant mechanisms have been evolved to several important herbicides. In this study we investigated the mechanisms responsible for conferring glyphosate resistance in some <i>Lolium</i> spp. populations. A holistic approach was used, based on dose-response experiments, determination of shikimic acid concentration in plant leaf tissue, as well as molecular analyses. More specifically, in three <i>Lolium</i> spp. populations the existence of a mutation in the Pro-106 codon of the 5-enolpyruvylshikimate-3 phosphate synthase (<i>EPSPS</i>) gene was investigated as well as the relative transcript levels of four <i>ABC-transporter</i> genes were monitored at three time points after glyphosate application. The results demonstrated that glyphosate resistance is a multifactor phenomenon. Relative transcript levels of the <i>ABC-transporter</i> genes were abundant at very early time points after glyphosate treatments. Dose-response experiments and shikimate analyses were in accordance with the findings of the quantitative PCR (qPCR) analyses. We suggest that relative expression ratio of <i>ABC-transporter</i> genes can be a useful tool to rapidly identify <i>Lolium</i> spp. populations resistant to glyphosate.