[Background] Endophyte from medicinal plants were important resource for discovery of new antibiotics due to their ability of producing a wide variety of secondary metabolites with good activity. Concerning the endophytic fungi, many studies have been carried out, but few reported about endophytic actinobacteria. [Objective] This study is aimed at analyzing the diversity of actinobacteria isolated from medicinal plants in Guizhou Province. [Methods] Various tissues from 8 kinds of medicinal plants as samples were collected in Guizhou Province, China. Samples were grinded into powders after surface sterilization and cultured on 9 different media for strain isolation. To determine the phylogenetic position of the strains, 16S rRNA gene sequences were obtained by PCR amplification and direct sequencing, then compared with EzBioCloud database, finally carried out phylogenetic analysis based on 16S rRNA gene sequences. [Results] A total of 62 strains were obtained, 57 of which were actinobacteria and were affiliated to 16 genera 8 families 5 order, contained genus Curtobacterium, Streptomyces, Microbacterium, Cellulomonas, Plantibacter, Promicromonospora, Kineococcus, Agrococcus, Patulibacter, Aeromicrobium, Labedella, Frondihabitans, Frigoribacterium, Arthrobacter, Kineosporia and Amnibacterium. The dominant genus is Curtobacterium. The highest similarity of 16S rRNA gene sequence between strain M8JJ-5 and the validly described species Amnibacterium kyonggiense KSL51201-037T (FJ527819), between strain M2KJ-4 and the validly described species Aeromicrobium fastidiosum DSM10552T (Z78209) was 97.29% and 98.95%, respectively, which indicated that the strains M8JJ-5 and M2KJ-4 were potential new taxa. [Conclusion] The study demonstrates that diversity of endophytic actinobacteria from medicinal plants are rich and medicinal plants as a special resource for discovery of new pharmaceutical microorganisms deserved to be further explored.