Herbicides are the most effective tools for controlling almost 99% of weeds. However, herbicide resistance is a primary concern in modern agriculture. The characterization in new areas and elucidation of the mechanisms of resistance are of vital importance in maintaining the sustainability of herbicides, including glyphosate. Nine populations of Lolium rigidum, showing different response patterns, were characterized as being glyphosate resistant (GR). The wide range of values in fresh weight reduction, survival, shikimic acid and EPSPS enzyme activity indicates a different or a combination resistance mechanism. The Line-3 population resulted in minimum reduction of fresh weight and survival values with respect to the glyphosate-susceptible (GS) population, showing 16.05- and 17.90-fold higher values, respectively. There were significant differences in the 14C-glyphosate translocation between GR and GS populations. Moreover, there were differences among the nine GR populations, but they exhibited a reduction in the remaining glyphosate translocation in the treated leaf. The EPSPS gene sequence revealed a Pro-106-Ser substitution in four populations, which could be characterized as being GR with non-target-site and target-site resistance mechanisms. This complexity of several resistance mechanisms makes it necessary to develop long-term integrated weed management strategies to limit further resistance dispersal.