Palmer amaranth (Amaranthus palmeri S. Watson) is one of the most problematic weeds in many cropping systems in the midsouthern United States because of its multiple weedy traits and its propensity to evolve resistance to many herbicides with different mechanisms of action. In Arkansas, A. palmeri has evolved metabolic resistance to S-metolachlor, compromising the effectiveness of an important weed management tool. Greenhouse studies were conducted to evaluate the differential response of A. palmeri accessions from three states (Arkansas, Mississippi, and Tennessee) to (1) assess the occurrence of resistance to S-metolachlor among A. palmeri populations, (2) evaluate the resistance level in selected accessions and their resistant progeny, (3) and determine the susceptibility of most resistant accessions to other soil-applied herbicides. Seeds were collected from 168 crop fields between 2017 and 2019. One hundred seeds per accession were planted in silt loam soil without herbicide for >20 yr and sprayed with the labeled rate of S-metolachlor (1,120 g ai ha−1). Six accessions (four from Arkansas and two from Mississippi) were classified resistant to S-metolachlor. The effective doses (LD50) to control the parent accessions ranged between 73 and 443 g ha−1, and those of F1 progeny of survivors were 73 to 577 g ha−1. The resistance level was generally greater among progeny of surviving plants than among resistant field populations. The resistant field populations required 2.2 to 7.0 times more S-metolachlor to reduce seedling emergence 50%, while the F1 of survivors needed up to 9.2 times more herbicide to reduce emergence 50% compared with the susceptible standard.