Plant small molecule UDP-glycosyltransferases (UGT) glycosylate a vast number of endogenous substances but also act in detoxification of metabolites produced by plant pathogenic microorganisms. The ability to inactivate the Fusarium graminearum mycotoxin deoxynivalenol (DON) into DON-3-O-glucoside is crucial for resistance of cereals. We analyzed the UGT gene family of the monocot model species Brachypodium distachyon and functionally characterized two gene clusters containing putative orthologs of previously identified DON-detoxification genes from Arabidopsis thaliana and barley. Analysis of transcription showed that UGTs encoded in both clusters are highly inducible by DON and expressed at much higher levels upon infection with a wild-type DON-producing F. graminearum strain compared to infection with a mutant deficient in DON production. Expression of these genes in a toxin sensitive strain of Saccharomyces cerevisiae revealed that only two B. distachyon UGTs encoded by members of a cluster of six genes homologous to the DON-inactivating barley HvUGT13248 were able to convert DON into DON-3-O-glucoside. Also, a single copy gene from Sorghum bicolor orthologous to this cluster and one of three putative orthologs of rice exhibit this ability. Seemingly, the UGT genes undergo rapid evolution and changes in copy number, making it difficult to identify orthologs with conserved substrate specificity.