Bovine tuberculosis (BTB) is an important neglected zoonosis that affects livestock, wildlife and human. A study to determine prevalence and geospatial clusters for BTB was conducted from June 2010 to March 2012 at livestock-wildlife interface areas (LWIA). A total of 1,288 cattle located in vicinity of Mikumi-Selous ecosystem Tanzania were tested. Single Intradermal Comparative Tuberculin Test and spatial scan statistic analysis were applied to establish the status of the disease and identify significant spatial BTB clusters. Overall individual prevalence was 3.7 % (n=1,288) (95 % CI=2.8-4.9) and 7.8 % (95 % CI=6.4-9.4) with cut-off of >4 and >2 mm, respectively. Villages with at least one reactor were 55.8 % (n=43). Reactivity was significantly higher in Mvomero and Kilosa districts compared with Kilombero and Ulanga districts (χ (2) =15.9; P<0.001). Significant spatial BTB clusters were revealed at 11 villages. BTB clustering was significant in Kilosa and Mvomero districts compared with Kilombero and Ulanga districts. There was overlap and aggregation of BTB clusters covering south and south-east of Kilosa district bordering Mikumi National Park (MNP) and Mvomero. Generally, clustering occurred around major rivers. The current study provides useful information on the dynamics and epidemiological status of BTB around the wildlife-livestock-human interface, it reveals that the wildlife are at risk of BTB from infected livestock. The study revealed hotspots for BTB that can be applied to guide implementation of participatory intervention at LWIA and control strategies in marginalised pastoralist communities. This study calls for similar studies in other Tanzania's LWIA for efficient intervention of BTB countrywide.