To establish a molecular epidemiological baseline for the strains causing tuberculosis in Nigeria, a survey of isolates from humans and cattle was carried out. Spoligotyping and variable-number tandem-repeat analysis revealed that the majority of tuberculosis disease in humans in Ibadan, southwestern Nigeria, is caused by a single, closely related group of Mycobacterium tuberculosis strains. Using deletion typing, we show that approximately 13% of the disease in humans in this sample was caused by strains of Mycobacterium africanum and Mycobacterium bovis rather than M. tuberculosis. Molecular analysis of strains of M. bovis recovered from Nigerian cattle show that they form a group of closely related strains that show similarity to strains from neighboring Cameroon. Surprisingly, the strains of M. bovis recovered from humans do not match the molecular type of the cattle strains, and possible reasons for this are discussed. This is the first molecular analysis of M. tuberculosis complex strains circulating among humans and cattle in Nigeria, the results of which have significant implications for disease control.