Palmer amaranth (Amaranthus palmeri S. Watson) is one of the most problematic weeds in U.S. agriculture, capable of rapidly adapting to environmental and management pressures. This study assessed temporal changes in glyphosate response in A. palmeri by comparing ED50 values, shikimic acid accumulation, and 14C-glyphosate absorption and translocation in four biotypes collected from two Georgia fields, Jones (J) and Little Jones (LJ), in 2008 and 2023. Glyphosate ED50 increased 9-fold (J08 vs. J23) and 25-fold (LJ08 vs. LJ23), indicating a marked reduction in glyphosate sensitivity between collection periods. Shikimic acid accumulation increased with glyphosate dose in all biotypes but remained substantially lower in biotypes collected in 2023, indicating reduced EPSPS inhibition. Radiolabeled assays revealed differences in early uptake, with populations collected in 2023 reaching near maximum absorption more rapidly, as reflected by shorter times to 95 percent absorption (A95), although total absorption continued to increase across all biotypes through 48 hours after treatment. Translocation patterns varied only slightly among biotypes, suggesting that changes in glyphosate response are associated more closely with altered uptake kinetics and EPSPS related mechanisms than with major reductions in systemic movement. These results demonstrate a temporal shift in glyphosate response in Georgia A. palmeri populations and highlight the importance of integrating kinetic analyses with traditional resistance metrics.