Greenhouse and laboratory studies were conducted to confirm and quantify glyphosate resistance, quantify pyrithiobac resistance, and investigate interaction between flumiclorac and glyphosate mixtures on control of Palmer amaranth from Mississippi. The GR50 (herbicide dose required to cause a 50% reduction in plant growth) values for two glyphosate-resistant biotypes, C1B1 and T4B1, and a glyphosate-susceptible (GS) biotype were 1.52, 1.3, and 0.09 kg ae ha−1 glyphosate, respectively. This indicated that the C1B1 and T4B1 biotypes were 17- and 14-fold resistant to glyphosate, respectively, compared with the GS biotype. The C1B1 and T4B1 biotypes were also resistant to pyrithiobac, an acetolactate synthase (ALS) inhibitor, with GR50 values of 0.06 and 0.07 kg ai ha−1, respectively. This indicated that the C1B1 and T4B1 biotypes were 7- and 8-fold, respectively, more resistant to pyrithiobac compared with the GS biotype, which had a GR50 value of 0.009 kg ha−1. Flumiclorac was antagonistic to glyphosate by reducing glyphosate translocation. The C1B1 and T4B1 absorbed less glyphosate 48 h after treatment (HAT) compared with the GS biotype. The majority of the translocated glyphosate accumulated in the shoot above the treated leaf (that contains the apical meristem) in the GS biotype and in the shoot below the treated leaf in the resistant biotypes, C1B1 and T4B1, by 48 HAT. The C1B1 biotype accumulated negligible shikimate levels, whereas the T4B1 and GS biotypes recorded elevated levels of shikimate. Metabolism of glyphosate to aminomethylphosphonic acid was not detected in either of the resistant biotypes or the susceptible GS biotype. The above results confirm multiple resistance to glyphosate and pyrithiobac in Palmer amaranth biotypes from Mississippi and indicate that resistance to glyphosate is partly due to reduced absorption and translocation of glyphosate.