genomic characterization of nontuberculous mycobacteria mycobacterium tuberculosis and mycobacterium leprae have remained for many years the primary species of the genus mycobacterium of clinical and microbiological interest the other members of the genus referred to as nontuberculous mycobacteria ntm have long been underinvestigated in the last decades however the number of reports linking various ntm species with human diseases has steadily increased and treatment difficulties have emerged despite the availability of whole genome sequencing technologies limited effort has been devoted to the genetic characterization of ntm species as a consequence the taxonomic and phylogenetic structure of the genus remains unsettled and genomic information is lacking to support the identification of these organisms in a clinical setting in this work we widen the knowledge of ntms by reconstructing and analyzing the genomes of 41 previously uncharacterized ntm species we provide the first comprehensive characterization of the genomic diversity of ntms and open new venues for the clinical identification of opportunistic pathogens from this genus