AbstractWaterhemp [Amaranthus tuberculatus (Moq.) Sauer] is a dioecious weed that has evolved resistance to very-long-chain fatty-acid elongase (VLCFAE)–inhibiting herbicides via rapid metabolism. Although detoxification enzyme activities are associated with S-metolachlor resistance in two multiple herbicide–resistant (MHR) A. tuberculatus populations from Illinois, the genetic basis of resistance is unknown. Therefore, our goal was to investigate inheritance of S-metolachlor resistance in the Stanford, Illinois–resistant (SIR) population. Specifically, our research objectives were to: (1) generate a uniformly resistant, full-sib near-inbred line (DK3-2) via three generations of recurrent selection for resistance using preemergence S-metolachlor; (2) develop A. tuberculatus populations segregating for S-metolachlor resistance via reciprocal single-plant (one male × one female) full-sib mating of DK3-2 and a VLCFAE-inhibiting herbicide-sensitive population, SEN; (3) quantify S-metolachlor resistance levels in parental lines and their F1 progenies via greenhouse dose–response analysis; and (4) evaluate inheritance of S-metolachlor resistance in F2 progenies. Dose–response analysis using six to eight S-metolachlor concentrations (0.015 to 15.0 μM, varying per population) generated lethal dose (LD) estimates of 50% (LD50) and 90% (LD90) for SIR, SEN, DK3-2, and F1 progenies. LD estimates indicated DK3-2 has a higher magnitude of S-metolachlor resistance than the SIR population, demonstrating single crosses significantly increased S-metolachlor resistance in DK3-2. Levels of S-metolachlor resistance in F1 populations were intermediate compared with DK3-2 and SEN. Segregation of S-metolachlor resistance in F2 families from the paternal-derived lines fit a single-gene model (R:S = 3:1), indicating a single, dominant gene confers S-metolachlor resistance in SIR. However, F2 segregation results from the maternal-derived lines fit a duplicate recessive epistasis model (R:S = 9:7), indicating a second recessive gene may also modify S-metolachlor resistance in SIR. Results and germplasm derived from this research can assist in identifying the gene(s) conferring resistance to S-metolachlor in A. tuberculatus.