Reactive carbonyl species， such as methylglyoxal and glyoxal are very toxic in nature and can inactivate various cellular macromolecules such as DNA， RNA， and protein by forming advanced glycation end products. Conventional glyoxalase pathway with two enzymes- glyoxalase I and glyoxalase II， detoxify MG into D-lactate with the help of reduced glutathione. However， DJ-1/PfpI domain(s) containing DJ-1/ Hsp31 proteins do the same in a single step， and thus termed as "glyoxalase III". A comprehensive genome-wide analysis of soybean identified eleven putative glyoxalase III proteins with DJ-1/PfpI domain encoded by seven genes. Most of these proteins are predicted to be mitochondria and chloroplast localized. In spite of similar function， a differential evolution pattern was observed between Hsp31 and DJ-1 proteins. Expression of GmDJ-1A， GmDJ-1B， and GmDJ-1D2 transcripts was found to be constitutive in different tissues and developmental stages. Transcript profiling revealed the strong substrate-specific upregulation of GmDJ-1 genes in response to exogenous methylglyoxal exposure. Out of seven genes， GmDJ-1D1 and GmDJ-1D2 showed maximum upregulation against salinity， dehydration， and oxidative stresses. Moreover， GmDJ-1D2 showed functional glyoxalase III enzyme activity by utilizing MG as a substrate. Overall， this study identifies some novel tissue-specific and abiotic stress-responsive GmDJ-1 genes that could be investigated further.