Almost complete sequences of the 16S rRNA genes of eight representative thermophilic streptomycetes were determined following the isolation and cloning of the amplified genes. These sequences were aligned with those of representative mesophilic streptomycetes, and phylogenetic trees were inferred by using four tree-making algorithms. The thermophilic streptomycetes formed two distinct clades that were supported by high bootstrap values based on 1,000 resamplings. One clade encompassed Streptomyces macrosporus and related species, and the second clade included Streptomyces thermodiastaticus and allied taxa. The relationships between the organisms were not markedly influenced by the different tree-making methods, but the rooting of the tree was sensitive to the choice of the outgroup strain. It is evident from this study that the thermophilic streptomycetes do not merit recognition as a distinct taxon within the genus Streptomyces.