In order to develop a gene clean deletion method in Thermus thermophilus without using selectable markers, the megaplasmid and the chromosome encoded TTP0042 and TTC0340_0341were chosen as target genes. The gene deletion vectors were flanked two homology target genes arms and then transformed into T. thermophilus HB27 cells with .the transformation mixtures streaked on selection free medium. The genotype of the resulted transformants were determined using PCR and Southern blot. The frequency of obtaining apparent Δ0042 mutant was 10–4 when using linearized TTP0042 gene deletion vector for transformation; while the frequency was 10–3 when used closed circular vector. The genotype and phenotype determination results showed that the apparent Δ0042 were all true TTP0042 gene clean deletion mutants. Therefore, in T. thermophilus, gene clean deletion mutants could be generated using the developed method with the frequency up to 10–3. The method was also verified in the chromosomal gene locus TTC0340_0341 and managed to generate the Δ0340_0341 mutant from 300 single colonies.