A line probe assay (GenoType MTBC) was evaluated for species differentiation within the Mycobacterium tuberculosis complex (MTBC). We included 387 MTBC isolates, 43 IS6110 low-copy MTBC isolates, 28 clinical specimens with varying microscopy grade, and 30 isolates of non-tuberculous mycobacteria. The assay was 100% specific and identified all 387 isolates and 98% of all IS6110 low-copy strains in concordance with the gold standard. The 2% discrepancy was caused by 1 isolate showing a faint restriction fragment length polymorphism (RFLP) pattern. The assay could provide specifies identification in 13 of 19 (68%) microscopy-positive specimens and 0 of 9 microscopy-negative specimens. To our surprise, the probe for M. africanum subtype I reacted with M. pinnipedii. This cross-reaction has not previously been reported. The assay was rapid, easy to perform and directly applicable in highly smear-positive specimens. We predict that the assay will enable enhanced surveillance of species-specific treatment outcome, which may change treatment regimens.