Objectives: Rapid species-specific identification is of clinical relevance since treatment varies according to the Mycobacterium species causing infection. The aim of this study was to evaluate the ability of Speed-oligo Mycobacteria (SpO-M) assay to correctly identify most frequently isolated Mycobacterium spp. cultured from clinical samples. Design: Comparative study Setting: Mycobacteriology Reference Laboratory, Department of Microbiology, Faculty of Medicine, Kuwait University, Kuwait Subjects: Mycobacterium species isolates (n=82) grown from human clinical samples in mycobacteria growth indicator tube (MGIT) 960 system and previously identified by molecular methods were tested. Interventions: DNA from liquid cultures was extracted, amplified by polymerase chain reaction (PCR), amplified products were detected by using the dipstick and results were interpreted according to SpO-M kit instructions. PCR-sequencing of 16S-23S internal transcribed spacer region of rDNA was used to confirm and validate SpO-M results. Main outcome measures: Concordance between SpO-M and original identification Results: The SpO-M correctly identified species/species complex for all Mycobacterium tuberculosis (n=34), and 45 of 48 nontuberculous mycobacteria. Mycobacterium lentiflavum was detected only as Mycobacterium species. One M. kansasii isolate was potentially misidentified as M. tuberculosis but a careful examination of SpO-M data resolved the discrepancy. Only one isolate, Mycobacterium parascrofulaceum, was misidentified as M. tuberculosis. Conclusions: Our data show that SpO-M is a rapid and reliable oligochromatographic test for detection and identification of most frequently isolated Mycobacterium species from clinical specimens in MGIT 960 system cultures for proper management of mycobacterial infections.