Fall panicum (Panicum dichotomiflorum Michx.) is an annual allochthonous grass weed currently spreading in maize fields in Spain. In the summer of 2022, poor control of P. dichotomiflorum with the acetolactate synthase (ALS)-inhibiting herbicide nicosulfuron was reported in two maize fields: Gerb, Catalonia (GB-R) and Sodeto, Aragon (SO-R), with a history of repeated nicosulfuron use. While target-site resistance (TSR) has previously been reported in this species, non–target-site resistance (NTSR) to ALS inhibitors has not yet been documented. The objectives of this study were to (1) confirm and quantify nicosulfuron resistance in the putative resistant populations GB-R and SO-R relative to the susceptible population TS-S; (2) characterize the presence of TSR mutations in the ALS gene; and (3) assess the potential involvement of NTSR via enhanced metabolism mediated by cytochrome P450 monooxygenases (P450s) and glutathione S-transferase (GSTs) enzymes. Dose–response assays confirmed that GB-R and SO-R populations were 29- and 37-fold more resistant than the TS-S population. However, co-application of nicosulfuron with the P450 inhibitor piperonyl butoxide (PBO) and the GST inhibitor 4-chloro-7-nitrobenzofurazan (NBD-Cl) significantly reduced resistance in both populations. In GB-R, GR50 values declined from 66.3 g ai ha–1 (nicosulfuron alone) to 15.8 with PBO and 6.5 with NBD-Cl, both well below the recommended field rate (40 g ha–1), corresponding to 76% and 90% resistance reductions, respectively. Similarly, in SO-R, GR50 values declined from 83.8 to 16.8 with PBO and 15.7 with NBD-Cl, representing 80% and 81% resistance reductions. Sequencing of the ALS gene revealed no known target-site mutations, and ALS enzyme activity did not significantly differ between R and S populations. These results suggest that nicosulfuron resistance in two Spanish P. dichotomiflorum populations is potentially associated with enhanced metabolism mediated by P450 and GST enzymes, providing the first worldwide evidence of NTSR in this species.