Context Available molecular epidemiological data from recent studies suggest significant genetic variation between the different lineages of Mycobacterium tuberculosis complex (MTBC) and the MTBC lineages might have adapted to different human populations. Aim This study sought to determine the population structure of clinical MTBC isolates from the Volta Region of Ghana. Methods The MTBC isolates obtained from collected sputum samples were identified by PCR detecting of IS6110 and genotyped using spoligotyping. Non-tuberculous mycobacterial isolates were characterized by amplification of the heat shock protein 65 (hsp65) gene and sequencing. The drug susceptibility profiles of the MTBCs determined using GenoType MTBDRplus. Results One hundred and seventeen (117, 93.6%) out of 125 mycobacterial positive isolates were characterized as members of the MTBC of which M. tuberculosis sensu stricto (MTBss) and M. africanum (MAF) were respectively 94 (80.3%) and 23 (19.7%). In all, 39 distinct spoligotype patterns were obtained; 26 for MTBss and 13 for MAF lineages. Spoligotyping identified 89 (76%) Lineage 4, 16 (13.6%) Lineage 5, 7 (6.0%) Lineage 6, 3 (2.6%) Lineage 2, 1(0.9%) Lineage 3 and 1 (0.9%) Lineage 1. Among the Lineage 4 isolates, 62/89 (69.7%) belonged to Cameroon sub-lineage, 13 (14.7%) Ghana, 8 (9.0%) Haarlem, 2 (2.2%) LAM, 1 (1.1%) Uganda I, 1 (1.1%) X and the remaining two (2.2%) were orphan. Significant localization of MAF was found within the Ho municipality (n = 13, 29.5%) compared to the more cosmopolitan Ketu-South/Aflao (n = 3, 8.3%) (p-value = 0.017). Eight (8) non-tuberculous mycobacteria were characterized as M. abscessus (7) and M. fortuitum (1). Conclusion We confirmed the importance of M. africanum lineages as a cause of TB in the Volta region of Ghana.