Reactive carbonyl species (RCS), such as methylglyoxal (MG) and glyoxal (GO), are synthesized as toxic metabolites in living systems. Mechanisms of RCS detoxification include the glutathione (GSH)-dependent system consisting of glyoxalase I (GLO1) and glyoxalase II (GLO2), and GSH-independent system involving glyoxalase III (GLO3). Hsp31 and DJ-1 proteins are weakly homologous to each other and belong to two different subfamilies of the DJ-1/Hsp31/PfpI superfamily. Recently, the Escherichia coli Hsp31 protein and the DJ-1 proteins from Arabidopsis thaliana and metazoans have been demonstrated to have GLO3 activity. We performed a systematic survey of homologs of DJ-1 and Hsp31 in fungi. We found that DJ-1 proteins have a very limited distribution in fungi, whereas Hsp31 proteins are widely distributed among different fungal groups. Phylogenetic analysis revealed that fungal and metazoan DJ-1 proteins and bacterial YajL proteins are most closely related and together form a sister clade to bacterial and fungal Hsp31 proteins. We showed that two Schizosaccharomyces pombe Hsp31 proteins (Hsp3101 and Hsp3102) and one Saccharomyces cerevisiae Hsp31 protein (ScHsp31) displayed significantly higher in vitro GLO3 activity than S. pombe DJ-1 (SpDJ-1). Overexpression of hsp3101, hsp3102 and ScHSP31 could confer MG and GO resistance on either wild-type S. pombe cells or GLO1 deletion of S. pombe. S. pombe DJ-1 and Hsp31 proteins exhibit different patterns of subcellular localization. Our results suggest that fungal Hsp31 proteins are the major GLO3 that may have some role in protecting cells from RCS toxicity in fungi. Our results also support the view that the GLO3 activity of Hsp31 proteins may have evolved independently from that of DJ-1 proteins.