In the present work, we investigated the ability of trpB gene, which encodes a primary metabolism enzyme involved in the tryptophan synthesis, to be used as an alternative to 16S rRNA for sequence similarity analysis in the Actinobacteria group. trpB DNAs (504 bp) were amplified from 13 Actinobacteria type strains, in addition to 24 environmental streptomycete isolates with different BOX-PCR profiles. The sequences and the phylogenetic tree of trpB were compared to those obtained from 16S rRNA gene analysis, for the total of the examined bacteria. The results demonstrated between 93 – 100 % (16S rRNA) and 86 – 100 % (trpB) similarity among the examined bacteria of the genus Streptomyces and suggested that trpB sequence similarity analysis allows a more accurate discrimination of the species within Streptomyces genus than the more commonly used 16S rRNA. Furthermore, DGGE analysis was also applied in habitats which exhibit a high degree of streptomycete diversity. The biodiversity patterns produced led to similar estimation of diversity, whether using 16S Actinobacteria group specific primers or the trpB novel primers. In conclusion, our study suggested that trpB sequence similarity analysis is a powerful tool for discrimination between species within the ecologically and industrially important strains of Streptomyces genus.