Abstract: Background and Objective: A comprehensive, phylogeny of genus Streptomyces is needed for a better understanding of their ecology as well as for facilitating their bioprospecting. 16S-rRNA-based phylogenetic reconstruction does not guarantee well-resolved and robust trees that reflect the overall relationship between Streptomyces species, therefore it is necessary to find a region of the genome that best shows the difference between Streptomyces. The goal of the present study was to produce a more robust phylogeny for Streptomyces by comparing the phylogenetic trees derived from concatenated gene and single gene sequence data. Methodology: Improvements in DNA sequencing technologies have resulted in the ability to generate large numbers of high quality draft genomes that have led to a dramatic increase in the number of publically available genomes and this has allowed researchers to characterize microorganisms using genomic data. In the present study, a phylogeny of 26 Streptomyces strains were analyzed using individual genes with more than 1 kb and compared with a phylogeny of 8 highly informative concatenated genes, for a total of 20 kb. Analyses were performed in MEGA, which defined the topology of the consensus tree. Results: The results from the concatenated genes showed a much higher power of discrimination and a much more stable topological structure than the 16S rRNA gene, with clearly better discriminated entities and higher bootstrap support. Comparing the 23S rRNA gene tree with the concatenated gene tree, it was found that the 23S rRNA tree had discriminatory power and topological stability similar to the concatenated gene tree. Conclusion: It is concluded that the 23S gene can be used as an alternative to 16S for the identification and classification of streptomycetes at species and intraspecies levels. The inner fragment of 23S (from 1 to 2 kb) is the most variable region and generated reliable and robust trees.