FK506 is an immunosuppressant discovered from culture of the streptomycete strain “Streptomyces tsukubaensis” 9993. Since its discovery, several streptomycete strains have been found as FK506 and/or ascomycin producers at our laboratory and other institutions. Ascomycin, which is also an immunosuppressant and anti-fungal macrolide, is structurally similar to FK506. We determined almost the full sequences of 16S rDNA of seven FK506- and/or ascomycin-producing streptomycete strains using direct PCR and a direct sequencing method. A homology search and phylogenetic analysis revealed that all strains belonged to the genus Streptomyces. The FK506-producing strains formed three clusters. The first cluster included Streptomyces sp. 6260 and 49A with “S. tsukubaensis” 9993, the second included Streptomyces sp. 94128 with Streptomyces kanamyceticus KCC S-0433^T, which was newly revealed to produce FK506 in this study, and the third included Streptomyces sp. ATCC 55098. The FK506-producing strains were phylogenetically suggested to belong to three different species. Furthermore, two ascomycin-producing strains phylogenetically differed from each other and the three FK506 producer clusters. The present findings confirmed that various phylogenetically different streptomycete strains produce FK506 and/or ascomycin. It is possible that the reason for this is the horizontal gene transfer of FK506 and ascomycin biosynthetic genes. Further phylogenetic investigation of these genes might therefore help further our understanding of the horizontal gene transfer and evolution of secondary metabolite genes.