Glyoxalase I (GLYI) is the key regulatory enzyme in the glyoxalase pathway. This pathway enables plants to neutralize methylglyoxal (MG) using glutathione (GSH), a mechanism significant for their acclimation to environmental stress. While functionally significant, the specific functions of GLYI genes in Amaranthus palmeri remain unexplored. In this study, integrated bioinformatics and expression analysis was used to identify five GLYI genes in A. palmeri. The results indicate that ApGLYI proteins are hydrophilic and slightly acidic, localized to scaffolds 1, 11, 13, and 16 of the A. palmeri genome. Phylogenetic analysis grouped ApGLYIs with other plant GLYI proteins into three distinct clades, each exhibiting conserved motif patterns. Expression analyses demonstrate that ApGLYI genes participate in both early and late regulatory phases of MG detoxification and signaling, responding to diverse stimuli including high temperature, NaCl, osmotic stress, exogenous methylglyoxal, abscisic acid (ABA), and methyl jasmonate (MeJA). Conversely, glufosinate ammonium treatment appears to compromise this cellular detoxification system. These results offer the evolutionary trajectory and functional significance of the ApGLYI gene. They establish a foundation for subsequent studies toward managing A. palmeri infestation and using these genes to improve stress resilience in cultivated crops through breeding strategies.