A study was carried out to determine the ALS (acetolactate synthase)-inhibitor herbicide resistance in the Mexican grass Ixophorus unisetus, a troublesome weed in corn crops in Mexico. First, the resistance was confirmed in field screening assays. Eight populations that survived nicosulfuron treatment at a field rate of 40 g ai ha−1 were labeled as putative-resistant. Dose–response trials demonstrated a high resistance in the eight populations (GR50 from 140.33 to 245.46 g ai ha−1). The synergism of malathion plus nicosulfuron demonstrated that the non-target-site resistance (NTSR) mechanism based on cytochrome P450 (Cyt-P450) was involved in five populations of I. unisetus. Molecular studies revealed that a single-nucleotide change occurs in the amino acid at position 376 (from GAT to GAG), which codifies from Asp-376 to Glu-376. This is the first time that Asp-376-Glu has been reported in this species. Assays in vitro and in vivo demonstrated I. unisetus cross-resistance to flucarbazone, penoxsulam, bispyribac-Na, and imazamox. No multiple resistance was found in two resistant populations exposed to different herbicides. Our results indicate that the lack of good control over Mexican grass in corn with ALS inhibitors is due to target-site mutation and NTSR mechanisms (Cyt-P450-mediated metabolism). A strategy should be established in Mexican fields to continue controlling this weed, including mechanical control practices and a good combination of the available pre- and post-emergence herbicides.