OBJECTIVES: While the reported incidence of non-tuberculous mycobacterial (NTM) infections is increasing, the true prevalence remains uncertain due to limitations in diagnostics and surveillance. The emergence of rare and novel species underscores the need for characterization to improve surveillance, detection, and management. METHODS: We performed whole-genome sequencing (WGS) and/or targeted deep-sequencing using the Deeplex Myc-TB assay on all NTM isolates collected in Slovakia and the Czech Republic between the years 2019 to 2023 that were unidentifiable at the species level by the routine diagnostic line probe assays (LPA) GenoType CM/AS and NTM-DR. Minimal inhibitory concentrations against amikacin, ciprofloxacin, moxifloxacin, clarithromycin, and linezolid were determined, and clinical data were collected. RESULTS: Twenty-eight cultures from different patients were included, of which 9 (32.1%) met the clinically relevant NTM disease criteria. The majority of those had pulmonary involvement, while two children presented with lymphadenitis. Antimycobacterial resistance rates were low. In total, 15 different NTM species were identified, predominantly rare NTM like M. neoaurum, M. kumamotonense and M. arupense. Notably, clinically relevant M. chimaera variants were also identified with WGS and Deeplex-Myc TB, which, unlike other M. chimaera strains, appeared to be undetectable by LPA assays. Deeplex detected four mixed infections that were missed by WGS analysis. In contrast, WGS identified two novel species, M. celatum and M. branderi, which were not detected by Deeplex-Myc TB. Importantly, one of these novel species strains was associated with clinically relevant pulmonary disease. DISCUSSION: Our study demonstrates the clinical relevance of uncommon NTM and the effectiveness of targeted deep-sequencing combined with WGS in identifying rare and novel NTM species.