Deinococcus radiodurans is extremely resistant to the effects of ionising radiation. The source of the radiation resistance is not known, but an expansion of specific protein families related to stress response and damage control has been observed. DNA-repair enzymes are among the expanded protein families in D. radiodurans, and genes encoding five different uracil-DNA glycosylases are identified in the genome. Here we report the three-dimensional structure of the mismatch specific uracil-DNA glycosylase (MUG) from D. radiodurans (drMUG) to a resolution of 1.75 Å. Structural analyses suggest that drMUG possesses a novel catalytic residue Asp93. Activity measurements show that drMUG has a modified and broadened substrate specificity compared to E. coli MUG. The importance of Asp93 for activity was confirmed by structural analysis and abolished activity for the mutant drMUGD93A. Two other micro-organisms, Bradyrhizobium japonicum and Rhodopseudomonas palustris, possess genes that encode MUGs with the highest sequence identity to drMUG among all bacterial MUGs examined. A phylogenetic analysis indicates that these three MUGs form a new MUG/TDG sub-family, here called the MUG2 family. We suggest that the novel catalytic residue (Asp93) has evolved to provide drMUG with broad substrate specificity in order to increase the DNA repair repertoire of D. radiodurans.