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Background: Non-Tuberculous Mycobacteria (NTM) are emerging around the world due to a higher prevalence of immunosuppressive illness and therapy. Saudi Arabia is not an exception as there have been novel mycobacterial species also identified. In addition, several published case reports from different parts of the country suggest a growing pathogenic potential of NTM. As the first nationwide study, we sought to gain an insight into the species diversity of NTM clinical isolates., Methodology/principal Findings: During June 2009-July 2010, 95 clinical isolates were collected from tuberculosis reference laboratories in major provinces within Saudi Arabia and subjected to standard line probe assay techniques to identify their species. Diagnostic guidelines of the American Thoracic Society were applied to determine the clinical relevance of respiratory isolates. Species diversity (13 species) was very high and dominated (61.0%) by rapid growing NTM. The major species obtained were Mycobacterium abscessus, M. fortuitum, M. intracellulare followed by M. kansassi, M. gordanae and M. avium. Interestingly this study reports for the first time the clinical relevance of M. celatum, M. xenopi, M. scrofulceum, M. lentiflavum, M. asiaticum and M. simiae in Saudi Arabia. Of the total, 67.1% were clinically relevant respiratory cases, 23.2% were non-respiratory cases and 9.7% were respiratory colonizers. Coexisting illness was reported in 53.7% of the studied cases. The major risk factors observed among the patients were previous history of tuberculosis, chronic obstructive pulmonary disorder and human immunodeficiency virus infection., Conclusion/significance: The high rates of clinically confirmed respiratory cases suggest that NTM infections are indeed a new challenge to health authorities. The current findings show an opposite picture of the Western world where M. avium complex and particularly slow growing NTM are the most predominant respiratory pathogens. The complexity of species demands an immediate strengthening of the current diagnostic facilities.