Populations of rigid ryegrass with resistance to glyphosate have started to become a problem on fence lines of cropping fields of southern Australian farms. Seed of rigid ryegrass plants that survived glyphosate application were collected from two fence line locations in Clare, South Australia. Dose–response experiments confirmed resistance of these fence line populations to glyphosate. Both populations required 9- to 15-fold higher glyphosate dose to achieve 50% mortality in comparison to a standard susceptible population. The mechanism of resistance in these populations was investigated. Sequencing a conserved region of the gene encoding 5-enolpyruvyl-shikimate-3-phosphate synthase identified no differences between the resistant and susceptible populations. Absorption of glyphosate into leaves of the resistant populations was not different from the susceptible population. However, the resistant plants retained significantly more herbicide in the treated leaf blades than did the susceptible plants. Conversely, susceptible plants translocated significantly more herbicide to the leaf sheaths and untreated leaves than the resistant plants. The differences in translocation pattern for glyphosate between the resistant and susceptible populations of rigid ryegrass suggest resistance is associated with altered translocation of glyphosate in the fence line populations.