Fourteen mycobacterial strains isolated from pulmonary samples of independent patients in the state of Pará (Brazil), and three strains isolated in Italy, were characterized using a polyphasic approach. Thorough genetic investigation, including whole-genome sequencing, demonstrated that the strains belong to the M. simiae complex, being most closely related to Mycobacterium interjectum . For 14 of the strains, evidence emerged supporting their inclusion in a previously unreported species of the genus Mycobacterium , for which the name Mycobacterium paraense sp. nov. is proposed (type strain, IEC26^T = DSM 46749^T = CCUG 66121^T). The novel species is characterized by slow growth, unpigmented or pale yellow scotochromogenic colonies, and a HPLC mycolic acid profile different from other known mycobacteria. In different genetic regions, high sequence microheterogeneity was detected.