In this study we characterized new strains of the slightly thermophilic species Rubrobacter radiotolerans and the thermophilic species Rubrobacter xylanophilus, both of which were previously represented only by the type strains isolated, respectively, from Japan and the United Kingdom. The new isolates were recovered from two hot springs in central Portugal after gamma irradiation of water and biofilm samples. We assessed biochemical characteristics, performed DNA-DNA hybridization, and carried out 16S rDNA sequence analysis to demonstrate that the new Rubrobacter isolates belong to the species R. radiotolerans and R. xylanophilus. We also show for the first time that the strains of R. xylanophilus and other strains of R. radiotolerans are extremely gamma radiation resistant.