Thermus thermophilus ( T . thermophilus ) HB27 is an extreme thermophile that grows optimally at 65–72 °C. Heat-induced DNA lesions are expected to occur at a higher frequency in the genome of T . thermophilus than in those of mesophiles; however, the mechanisms underlying the maintenance of genome integrity at high temperatures remain poorly understood. The study of mutation spectra has become a powerful approach to understanding the molecular mechanisms responsible for DNA repair and mutagenesis in mesophilic species. Therefore, we developed a supF -based system to detect a broad spectrum of mutations in T . thermophilus . This system was validated by measuring spontaneous mutations in the wild type and a udgA , B double mutant deficient in uracil-DNA glycosylase (UDG) activity. We found that the mutation frequency of the udgA , B strain was 4.7-fold higher than that of the wild type and G:C→A:T transitions dominated, which was the most reasonable for the mutator phenotype associated with the loss of UDG function in T . thermophilus . These results show that this system allowed for the rapid analysis of mutations in T . thermophilus , and may be useful for studying the molecular mechanisms responsible for DNA repair and mutagenesis in this extreme thermophile.