The WHO reported in 1998 that 3.1% of tuberculosis cases in humans worldwide are attributable to M. bovis and that in 0.4-10% of sputum isolates from patients in African countries, M. bovis is isolated. Sputum samples were collected from a total of 917 smear-positive TB patients enrolled in a national drug resistance survey in the nine provinces of Zambia and another 100 patients enrolled in a separate TB survey conducted in the pastoral area of Namwala district of Southern province of Zambia between 2008 and 2011. Based on Spoligotyping, eight of the isolates from both surveys were confirmed as M. bovis belonging to the SB 0120 Spoligotype. The two surveys provided an opportunity to document isolation of M. bovis from sputum samples from patients diagnosed with TB from both urban and pastoral areas of Zambia. This study therefore, highlights the public health significance of M. bovis in Zambia and the importance of screening for M. bovis as part of routine diagnosis procedures. Hence, a targeted treatment for those human patients suffering from zoonotic tuberculosis is recommended to address important differences in pathology and treatment response between different mycobateria.