Abiotic stresses have deleterious effects on seed germination and seedling establishment, leading to significant crop yield losses. Adverse environmental conditions can cause the accumulation of methylglyoxal (MG) within plant cells, which can negatively impact plant growth and development. The glyoxalase system, which consists of the glutathione (GSH)-dependent enzymes glyoxalase I (GLX1) and glyoxalase II (GLX2), as well as the GSH-independent glyoxalase III (GLX3 or DJ-1), plays a crucial role in detoxifying MG. However, genome-wide analysis of glyoxalase genes has not been performed for one of the agricultural important species, oat (Avena sativa). This study identified a total of 26AsGLX1genes, including 8 genes encoding Ni2+-dependent GLX1s and 2 genes encoding Zn2+-dependent GLX1s. Additionally, 14AsGLX2genes were identified, of which 3 genes encoded proteins with both lactamase B and hydroxyacylglutathione hydrolase C-terminal domains and potential catalytic activity, and 15AsGLX3genes encoding proteins containing double DJ-1 domains. The domain architecture of the three gene families strongly correlates with the clades observed in the phylogenetic trees. TheAsGLX1,AsGLX2, andAsGLX3genes were evenly distributed in the A, C, and D subgenomes, and gene duplication ofAsGLX1andAsGLX3genes resulted from tandem duplications. Besides the core cis-elements, hormone responsive elements dominated the promoter regions of the glyoxalase genes, and stress responsive elements were also frequently observed. The subcellular localization of glyoxalases was predicted to be primarily in the cytoplasm, chloroplasts, and mitochondria, with a few presents in the nucleus, which is consistent with their tissue-specific expression. The highest expression levels were observed in leaves and seeds, indicating that these genes may play important roles in maintaining leaf function and ensuring seed vigor. Moreover, based on in silico predication and expression pattern analysis,AsGLX1-7A,AsGLX2-5D,AsDJ-1-5D,AsGLX1-3D2, andAsGLX1-2Awere suggested as promising candidate genes for improving stress resistance or seed vigor in oat. Overall, the identification and analysis of the glyoxalase gene families in this study can provide new strategies for improving oat stress resistance and seed vigor.