The family Streptomycetaceae, notably species in the genus Streptomyces, have long been the subject of investigation due to their well-known ability to produce secondary metabolites. The emergence of drug resistant pathogens and the relative ease of producing genome sequences has renewed the importance of Streptomyces as producers of new natural products and resulted in revived efforts in isolating and describing strains from novel environments. A previous large study of the phylogeny in the Streptomycetaceae based on 16S rRNA gene sequences provided a useful framework for the relationships among species, but did not always have sufficient resolution to provide definitive identification. Multi-locus sequence analysis of 5 house-keeping genes has been shown to provide improved taxonomic resolution of Streptomyces species in a number of previous reports so a comprehensive study was undertaken to evaluate evolutionary relationships among species within the family Streptomycetaceae where type strains are available in the ARS Culture Collection or genome sequences are available in GenBank. The results of the analysis supported the distinctiveness of Kitasatospora and Streptacidiphilus as validly named genera since they cluster outside of the phylogenetic radiation of the genus Streptomyces. There is also support for the transfer of a number of Streptomyces species to the genus Kitasatospora as well for reducing at least 31 species clusters to a single taxon. The multi-locus sequence database resulting from the study is a useful tool for identification of new isolates and the phylogenetic analysis presented also provides a road map for planning future genome sequencing efforts in the Streptomycetaceae.