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We present the first evaluation of a novel molecular assay, the Speed-Oligo Mycobacterium tuberculosis complex (SO-MTBC), which is based on PCR combined with a dipstick for the differentiation of M. tuberculosis complex (MTBC) members. The results of this assay were compared with findings obtained using the Genotype MTBC assay. In this study, 189 strains of MTBC isolates from 2011 to 2014 were evaluated to determine the MTBC species. Most (174, 92 %) of the strains were identified as M. tuberculosis sensu stricto, 7 (3.7 %) as Mycobacterium bovis, 5 (2.6 %) as M. bovis bacillus Calmette-Guérin, 2 (1.1 %) as Mycobacterium africanum and 1 (0.5 %) as Mycobacterium caprae; no strains belonged to Mycobacterium microti and Mycobacterium canettii subsp. The concordance κ coefficient obtained was 0.96 with the results of the Genotype MTBC assay. SO-MTBC may represent a fast and easy-to-use alternative for differentiating among MTBC subspecies in laboratories with standard equipment.