Four species of non-tuberculous mycobacteria (NTM) rated as biosafety level 1 or 2 (BSL-1/BSL-2) organisms and showing higher genomic similarity with () than previous comparator species and were subjected to genomic and phenotypic characterization. These species named , , and might represent "missing links" between low-virulent mycobacterial opportunists and the highly virulent obligate pathogen . We confirmed that is the closest NTM species to currently known and found that it has an optimal growth temperature of 32°C-35°C and not 37°C. showed resistance to rifampicin, isoniazid, and ethambutol, whereas and showed resistance to isoniazid and ethambutol. was sensitive to all three first-line TB drugs, and all four species were sensitive to bedaquiline, a third-generation anti-TB drug. Our results suggest these four NTM may be useful models for the identification and study of new anti-TB molecules, facilitated by their culture under non-BSL-3 conditions as compared to was the most virulent of the four species in cellular and mouse infection models. also multiplied in THP-1 cells at 35°C but was growth impaired at 37°C. Genomic comparisons showed that the locus, essential for the secretion of ESX-1 proteins in , was present only in , which was able to secrete ESAT-6 and CFP-10, whereas secretion of these antigens varied in the other species, making the four species interesting examples for studying ESX-1 secretion mechanisms.IMPORTANCEIn this work, we investigated recently identified opportunistic mycobacterial pathogens that are genomically more closely related to () than previously used comparator species and . We confirmed that is the currently closest known species to the tubercle bacilli, represented by and strains. Surprisingly, the reference strain of (DSM 45176), which was purchased as a biosafety level 1 (BSL-1)-rated organism, was the most virulent of the four species in the tested cellular and mouse infection models, suggesting that a BSL-2 rating might be more appropriate for this strain than the current BSL-1 rating. Our work establishes the four NTM species as interesting study models to obtain new insights into the evolutionary mechanisms and phenotypic particularities of mycobacterial pathogens that likely have also impacted the evolution of the key pathogen .