AbstractPalmer amaranth (Amaranthus palmeri S. Watson) is not native to Africa. Based on the presence and persistence of A. palmeri populations, its invasive status in southern Africa is classified as “naturalized.” Globally, A. palmeri is one of the most troublesome weed species in several crops, including soybean [Glycine max (L.) Merr.], maize (Zea mays L.), and cotton (Gossypium hirsutum L.). Certain populations of A. palmeri in various countries were reported to be resistant to herbicides with different sites of action (SOAs). Two biotypes of A. palmeri in the United States reportedly each have resistance to herbicides representing five different SOAs, and between them a total of eight different SOAs are involved. Resistance mechanisms in these biotypes involve target-site and/or non–target site resistance. Here we characterize a specific A. palmeri population that was found in the Douglas district in South Africa and showed resistance to various herbicide SOAs. Initially, this A. palmeri population was discovered in a glyphosate-tolerant cotton field, where it survived glyphosate treatment. Subsequently, greenhouse experiments were conducted to characterize this A. palmeri population for potential resistance to herbicides of additional SOAs, and molecular analyses were conducted to reveal the mechanisms of herbicide resistance. Results indicated resistance to chlorimuron-ethyl and glyphosate in this population, while <90% control (decreased sensitivity) was observed at the label rate for mesotrione, atrazine, saflufenacil, and S-metolachlor. However, glufosinate, tembotrione, acifluorfen, dicamba, 2,4-D, metribuzin, acetochlor, isoxaflutole, diflufenican, and pyroxasulfone were effective at controlling this population. This profiling of herbicide sensitivity has allowed development of programs to control and potentially minimize the spread of this weed. In addition, molecular analysis of EPSPS revealed the role of higher copy number as a mechanism for glyphosate resistance in this population and a Ser-653-Asn target-site mutation likely conferring resistance to the acetolactate synthase–inhibitor chlorimuron-ethyl. No known target-site mutations were identified for the protoporphyrinogen oxidase–inhibitor group.