We have identified an active insertion sequence (IS) in the genome of Thermus thermophilus HB8. Transposition was detected as insertional inactivation of a 16S rRNA methyltransferase gene, rsmG, resulting in streptomycin resistance. The IS element, ISTth7, is 1029 bp in length, encodes an imperfect 12 bp inverted repeat, and produces a 9 bp direct repeat of the target sequence. The sequence of a putative transposase encoded by ISTth7 indicates that it is a member of the IS427 group within the IS5 family of ISs. Nine intact copies and several partial copies were identified throughout the chromosome and the megaplasmid pTT27. ISTth7 was also detected in T. thermophilus strain IB-21 and Thermus igniterrae but not Thermus antranikianii, suggesting a widespread occurrence of ISTth7 among Thermus spp.