Objective: Characterize mycobacterial species causing pulmonary tuberculosis (PTB) at the Korle-Bu Teaching Hospital in Ghana. Design: Sputum smear positive samples, two (2) from 70 patients diagnosed as having tuberculosis, after they had consented, were collected from the Korle-Bu Teaching Hospital Chest Clinic between January and July 2003. Setting: Korle-Bu Teaching Hospital Chest Clinic, Accra. Results: Sixty-four mycobacterial isolates were obtained and confirmed as members of Mycobacterium tuberculosis complex by colonial morphology and conventional biochemical assays. Fortyseven (73%) were M. tuberculosis, the human strain, 2 (3%) M. bovis, the bovine strain, 13 (20%) M. africanum I (West Africa type), and 2 (3%) M. africanum II (East Africa type). Conclusion: The results indicate that, there are various strains causing PTB at the Korle-Bu Teaching Hospital and of great concern is M. bovis, which mostly causes extra-PTB in humans but found to cause PTB in this study. This calls for the need to conduct a nationwide survey using both conventional and molecular techniques to characterize various mycobacterial species causing TB in Ghana. This will result in better understanding of the various strains circulating in the country and inform individual TB treatment regimen especially the inclusion or exclusion of pyrazinamide.