Novel biomarkers are urgently needed for point of care TB diagnostics. In this study, we investigated the potential of the pilin subunit protein encoded by the mtp gene as a diagnostic biomarker. BLAST analysis of the mtp gene on published genome databases, and amplicon sequencing were performed in Mycobacterium tuberculosis Complex (MTBC) strains and other organisms. The protein secondary structure of the amino acid sequences of non-tuberculous Mycobacteria that partially aligned with the mtp sequence was analysed with PredictProtein software. The mtp gene and corresponding amino acid sequence of MTBC were 100% homologous with H37Rv, in contrast to the partial alignment of the non-tuberculous Mycobacteria. The mtp gene was present in all 91 clinical isolates of MTBC. Except for 2 strains with point mutations, the sequence was 100% conserved among the clinical strains. The mtp gene could not be amplified in all non-tuberculous Mycobacteria and respiratory organisms. The predicted MTP protein structure of Mycobacterium avium, Mycobacterium ulcerans and Mycobacterium abscessus differed significantly from that of the M. tuberculosis, which was similar to Mycobacterium marinum. The absence of the mtp gene in non-tuberculous Mycobacteria and other respiratory bacteria suggests that its encoded product, the pilin subunit protein of M. tuberculosis may be a suitable marker for a point of care TB test.