The article presents data on the structure of acid-resistant members of the order Actinomycetales and rare species that have been isolated and identified using various methods. The study included strains of non-tuberculous mycobacteria (NTM) isolated from clinical material during examination for tuberculosis in the period from 2016 to 2019. The total number of samples with signs of NTMs growth that were included in the study was 316 samples. Primary isolation on Levenshtein-Jensen, Finn II, and MGIT media and NTMs identification by DNA-hybridization. All strains that were not identified prior to the species and culture, identified as microorganisms with a high G+C content (High GC GR +) were re-identified using a MALDI-ToF Microflex LT mass spectrometer (Bruker®). By the method of DNA-hybridization, 188 strains isolated by NTM were successfully identified to form 58.5% of all selected cultures. Among the selected species, representatives of slowly growing NTMs (M. avium complex, M. gordonae, M. kansasii) predominated, which amounted to 67.0% of all NTM strains identified to the species. Among the cultures for which DNA hybridization failed to carry out acceptable identification, predominantly NTMs were found, among which M. gordonae, M. avium, M. kansasii dominated. A number of NTMs were represented by rare species: M. iranicum and M. pseudoshottsii. Among this group of microorganisms, other acid-resistant aerobic actinomycetes were isolated, including those of potential clinical significance: Gordonia spp., Tsukamurella spp., Rhodococcus spp., Nocardia spp. When identifying cultures containing high concentrations of G+C, the maximum number of microbial associations was revealed, including those consisting of two types of NTMs (M. monacense + M. flavescens, M. avium + M. kansasii), as well as associations of M. gordonae with staphylococci. The same group included rare NTM species: M. fredericbergense, M. szulgai, M. malmoense, M. bohemicum, M. septicum, as well as representatives of the genera Nocardia, Gordonia, Tsukamurella.