Common ragweed is a weed in the midwestern United States and eastern Canada that is difficult to control due to the evolution of an important resistance to multiple herbicides including glyphosate. Recently, a common ragweed biotype with 19-fold glyphosate resistance was confirmed in Nebraska. The objective of this study was to determine the mechanism of glyphosate resistance in a common ragweed biotype from Nebraska. Both target site and non-target site based mechanisms of glyphosate resistance were investigated using glyphosate-resistant (GR) and known glyphosate-susceptible (GS) common ragweed biotypes. A lower amount of shikimate was accumulated in the GR (≤65 µg mL^-1) compared with the GS (≥80 µg mL^-1) biotype at all glyphosate concentrations tested. Sequencing of the conserved region of the EPSPS gene revealed no mutations at the Thr₁₀₂ or Pro₁₀₆ residues and no variation in EPSPS copy number was detected. A higher translocation of ¹⁴C-glyphosate in the GR compared with the GS biotype was found, although there was no difference in the amount of ¹⁴C-glyphosate absorbed. Nonetheless, analysis of ¹⁴C-glyphosate absorption or translocation data using the rectangular hyperbolic model predicted a slower rate of absorption and translocation of glyphosate in the GR compared with the GS biotype, though more research is needed. These results indicate possible involvement of a non-target site mechanism bestowing resistance to glyphosate. The possibility that a slow rate of glyphosate absorption and translocation might have a role in preventing the buildup of the minimum inhibitory concentration of glyphosate required at the target site needs further research.