Actinomycetes are important producers of bio-active metabolites, including antibiotics, plant growth factors, enzyme inhibitors and many other beneficial compounds. Alpine extreme habitats that contain low temperature-, anoxia-, and stress-adapted plants, and rhizosphere soils may harbor novel actinomycete strains with unique metabolic characteristics and new secondary metabolites. The Laohugou Valley of the Qilian Mountains, in the northeastern of the Tibetan Plateau, is characterized by its high elevation (> 2000 m) and a distinct vertical distribution of vegetation types, ranging among desert steppe at 2200 m, mesophorbium at 2800 m, alpine shrub at 3350 m, alpine meadow at 3800 m, and alpine cold-desert at 4200 m. However, little is known about the diversity and distribution of actinomycetes in these habitats. Thus, we analyzed 15 rhizosphere soil samples from different vegetation types at increasing altitudes in Laohugou Valley to investigate the diversity and physiological characteristics of actinomycetes. We recovered 78 strains of actinomycetes with representative phenotype using glycerin arginine medium with potassium dichromate and penicillin. The morphology of colony hypha, small subunit 16S rRNA sequences, phylogenetic relationships, and physiological heterogeneity of these isolates were determined. The results revealed that 73 strains were closely related to Streptomyces spp. (93.6% of all isolates), which grouped into 21 species. Streptomyces spp. were found in all samples, where the abundance and diversity varied at different altitudes among rhizosphere soils. The highest diversity of Streptomyces spp. was found in the rhizosphere soil of Astragalus licentianu at 4200 m and the lowest in the rhizosphere soil of Poa annua at 3800 m. Four isolates (5.1% of all isolates) in the genus Nocardia were found in the rhizosphere soils of Salsola collina at 2200 m, Potentilla saudersiana at 2800 m and Arenaria kansuensis at 3800 m. We also isolated a strain from the rhizosphere soil of Stipa glareosa at 2200 m which shared 92% sequence similarity to its nearest match Micromonospora saelicesensis from database, indicating that it might be a new actinomyces species. The physiological analyses of the isolates indicated that strains producing catalase, lipase 2 (Tween-40), urease, proteinase, lipase 3 (Tween-80), amylase, H₂S, lipase 1 (Tween-20), diffusible pigment and organic acids accounted for 89.7%, 82.1%, 70.5%, 62.8%, 53.8%, 52.6%, 48.7%, 44.9%, 32.1% and 17.9% of the total actinomycetes isolates, respectively. Amylase, lipase 1, pigments and organic acid were produced only by Streptomyces isolates. The antimicrobial resistant patterns of the isolates were tested with Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Candida albicans (ATCC 66415), and a Pseudomonas aeruginosa strain isolated from clinical sputum. The results showed that 29 strains isolated from the rhizosphere soils of 12 plants at different altitudes exhibited antimicrobial activity, i.e., 37.2% of the total isolates. The isolates from the rhizosphere soils of two medicinal plants (Astragalus licentianu and Rhodiola quadrifida) comprised 60% of the total antimicrobial strains. The distinct antibiotic resistant patterns and physiological traits were detected among strains within the identical species. Our results suggested that the rhizosphere soils of alpine plants in the Qilian Mountain may be potential sources of new actinomycetes strains with novel bioactive compounds.