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Tuberculosis (TB) is one of the infectious diseases with high mortality rate in the world. It is highly endemic among third-world countries with large prevalence in Asia and sub-Saharan Africa. In Ghana, there is a mixed population of mycobacteria strains causing TB namely; M. tuberculosis, M. africanum and M. bovis. Some of the major problems confronting TB management include; lack of rapid, reliable and robust method of diagnosing and differentiating mycobacterial tuberculosis complex (MTBC) species, inherent drug resistance of some species and co-morbidity with HIV, etc. Hence, the objective of this study is to diagnostically identify and differentiate MTBC species causing TB in Ghana. Genome sequences of M. tuberculosis, M. africanum and M. bovis were obtained from NCBI and EMBL-EBI repositories for pangenome analysis. The Mauve alignment of complete genomes was used to check for the uniqueness of genes. Candidate genes were tested by BLAST against the genome sequences of all M. tuberculosis, M. africanum and M. bovis to check for their presence or absence. Three (3) genes were identified to be specific to M. tuberculosis: Rv1977, Rv2073c and Rv2074. The PPE domain Protein (MAF263_35110) and beta-glucosidase (MAFGCA_01990) genes were identified to be unique to M. africanum lineages L5 and L6/ L9 respectively. The Rv3903c gene was identified to be present in all the mycobacterial genomes and hence, could be used for designing a positive control primer. There was no unique gene for M. bovis due to deletions of large genomic regions. However, the formation of a band by only the positive control primer during a multiplex PCR reaction involving all the other primers, could indicate the presence of M. bovis. For multiplex PCR assay, primers were designed for all the genes by assigning specific amplicon size to each of them using the PrimerQuest Tool. In low resource settings with high incidence of tuberculosis, differentiation of MTBC species using a multiplex PCR assay would provide rapid and reliable method of disease diagnosis, improve epidemiological surveillance and help inform appropriate TB drug selection at early time point.&nbsp