Background Non-tuberculous mycobacteria (NTM) are ubiquitous pathogens in opportunistic infection. Because the types of NTM existing in the living environment are diverse, it is expected that NTM patients will be exposed to new types of NTM bacteria every day. Therefore, cases of mixed infection in non-tuberculous mycobacterium lung disease are very common, and it has already been known that the statistical significance between mixed infection and low treatment success rate. In this study, we confirmed the possibility of the mixed infection among heterogeneous or subspecies-level through various phenotypes in NTM clinical isolates. Methods Total 1,095 clinical isolates of NTM collected by Korea Mycobacterium Resource Center from March 2020 to June 2021 were used. Identification of the species was performed through Mycobacteria Genoblot Assay by extracting DNA of cultured strains. Confirmation of the morphological diversity was observed by cultured colony shapes inoculating strains through three-phase streaking pattern on 7H10 agar medium. Results A total of 1095 clinical strains were identified, and that Results were as follows: 397 M. avium, 397 M. intracellulare, 121 M. abscessus, 62 M. massiliense, 20 M. fortuitum, 43 rare species, and 55 Mixed species. Excluding mixed species, 122 M. avium, 262 M. intracellulare, 9 M. abscessus, 2 M. massiliense, 7 M. fortuitum and 3 rare species had multiple phenotypic colonies in one clinical isolate, respectively. Conclusion Mixed infection through molecular diagnosis was confirmed in 55 out of 1095, but cases of 405 had phenotypic diversity among clinical isolates confirmed as a single species. It suggests that additional mixed infections not confirmed by molecular diagnosis is may exist at the species-level or subspecies-level, or that a phenotypic transition may occur. In order to observe the correct treatment response and the prognosis for NTM disease patients, it is necessary to confirm such a mixed infection or phenotypic transition.