The relevance of the use of molecular tools in the global epidemiology of Mycobacterium tuberculosis complex (MTBC) cannot be undermined. Molecular epidemiological studies of the MTBC in Nigeria are not extensive, and to date, there has only been one detailed report. More strains are therefore needed to be genotyped in order to give a clear indication of disease transmission chains and to highlight routes of infection particularly with respect to zoonotic tuberculosis. This study therefore focuses on the identification and genotyping of MTBC isolates in south western Nigeria, with emphasis on interactions occurring at the human/livestock interface. The molecular epidemiology of M. bovis strains in Hluhluwe-iMfolozi Park in South Africa was also undertaken. Prior to this study, a pilot study was initially done to establish techniques, using samples from Belgium. Mycobacterium bovis strains were first identified in Belgium using the Multiple locus [variable number of tandem repeats] (MLVA) technique and analysis was done using capillary electrophoresis. In this study, the Belgium isolates were repeated using MLVA and analysis by agaorse gel electrophoresis and the two analysis techniques compared. Human isolates (136) and livestock isolates from cattle (50), pigs (12) and goats (5) isolated in Nigeria were also used and species identification of the members of the MTBC were done using the deletion analysis PCR technique amplifying RD1mic, RD2seal, RD4 andRD9 regions as well as spoligotyping. Seventy four positive MTBC strains (humans and livestock) were genotyped using 16 VNTR loci. The discriminatory ability of the 16 loci MLVA was compared with spoligotype data on 33 MTBC strains. Mycobacterium bovis isolates from buffalo in HluhluweiMfolozi Park (HiP) South Africa, were also genotyped using the 16 loci MLVA and spoligotyping. Results indicated that agarose based MLVA is as discriminatory as the capillary based MLVA. Furthermore, the relevance of molecular techniques in the rapid identification and genotyping of members of the MTBC, especially in a tuberculosis endemic setting like Nigeria, is also highlighted. This was clearly seen in the identification of undescribed spoligopatterns of the LAM 10-CAM M. tuberculosis strains in humans as well as the identification of undescribed M. bovis spoligopatterns in livestock isolates. The prevalent M. bovis strain (SB0944) in Nigeria was also identified in a human isolate. Also, two classical M. bovis strains were identified in two human isolates obtained from cattle traders, thus suggesting the influence of close interaction between infected animals and man as a means of zoonotic tuberculosis transmission. Mycobacterium tuberculosis was also identified in three isolates, from cattle, pig and goat; with the goat isolate having a spoligopattern (EAI5) typical of strains indigenous to East Africa and India. This study demonstrated the prevalent strains of M. bovis and M. tuberculosis circulating in Nigeria with SB0944 the predominant M. bovis spoligotype and LAM10-CAM the predominant M. tuberculosis spoligotype. The MLVA results revealed the occurrence of interspecies transmission of mycobacterial species, which was seen as isolates from different animal species having identical VNTR profiles and thus belonging to the same genotype. In the HiP, two strains of M. bovis were identified, a strain previously described in cattle and buffalo in other regions of South Africa and a new undescribed strain, thus giving an indication of the circulating strains in HiP and also suggesting possible sources of introduction of novel species in HiP. The relevance of a detailed molecular epidemiological study was clearly demonstrated in both Nigeria and HiP. Strain relatedness and interactions occurring at human/livestock interface and domestic/wild life interface could also clearly be demonstrated.