Journal of Agricultural Science and Technology B 2014, Vol. 4 215-223A study on nontuberculous mycobacteria (NTM) was carried out in wildlife-livestock interface of Katavi Rukwa ecosystem (KRE). 328 livestock tissues and 178 wild animals were cultured, wild animals were sampled opportunistically during professional hunting and game cropping operations in the KRE protected areas. The objective of the study was to generate data on epidemiology of NTM in the wildlife-livestock interface of the KRE. Methods used to identify the NTM were: culture and isolation, polymerase chain reaction, protein heat shock 65 kilodalton (hsp65) and sequencing. Mycobacteria were detected on 25.9% and 11.9% of livestock and wildlife tissue cultures, respectively. The most NTM isolated were M. kansasii (30%), M. gastri (30%), M. fortuitum (1%), M. intracellulare (4%), M. indicus pranii (4%), M. nonchromogenicum (6%) and M. lentiflavum (6%). Other NTM in smaller percentages were M. hibernae, M. engbaekii, M. septicum, M. arupense and M. godii. Due to rise of NTM infection in both human and animals, it is recommended that awareness and laboratory facilities be improved to curb the underreporting especially in TB-endemic countries. For species specific identification, a network of national and regional laboratories is promoted.