[Objective] The objective of this study was to apply multilocus sequence analysis (MLSA) to evaluate the phylogenetic relationship among highly similar strains of the genus Streptomyces isolated from the Indian Ocean sediment samples under the depth of 3 000 m, based on 16S rRNA, atpD, recA and rpoB genes, and to further explore the potential of isolated Streptomyces species. [Methods] We cultured 7 strains of Streptomyces albidoflavus, 11 strains of Streptomyces cavourensis, 16 strains of Streptomyces pratensis isolated from the Indian Ocean deep-sea sediments. We used PCR amplification and sequenced the genes 16S rRNA, atpD, recA and rpoB from these strains and also downloaded 5 strains sequences of S. pratensis from NCBI. We used all sequences to find the gene types in MLST web site and to build the phylogenetic trees for comparison. [Results] After comparison of S. pratensis, the same genotype strains were not clustered together in phylogenetic tree based on 16S rRNA gene sequence, the tree was not stable and degree of differentiation was also low, the phylogenetic trees based on atpD, recA, rpoB genes were relatively stable. Strains in the phylogenetic tree based on multiple gene cluster analysis divided into six groups. In Multilocus sequence analysis of the three Streptomyces, the degree of differentiation increased and the phylogenetic tree was more stable and also found some special strains of recA gene. [Conclusion] Multilocus sequence analysis can divide experimental strains into different types and some strains have large differences in individual gene (recA gene). Multilocus sequence analysis is suitable for classification and identification of some closely related species.