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Background and Aim: Bovine tuberculosis (bTB) remains a major public health issue in Zambia and has been exacerbated by human immunodeficiency virus prevalence and consumption of unpasteurized milk in the Southern Province of the country. The prevalence of bTB has been established to be linked to Kafue Lechwe, which act as reservoir hosts and share grazing fields with domestic cattle. No studies have so far used geographic information system (GIS) to investigate the relationship between the reservoir hosts (Kafue Lechwe) and domestic animals. This study, therefore, aimed to apply GIS to investigate the spatial distribution of bTB in Namwala District of the Southern Province of the country., Materials and Methods: To investigate the spatial distribution of bTB, geographical positioning system (GPS) coordinates representing 96 cattle herds across 20 independent villages were captured alongside risk factor data. The 96 herds were based on abattoir reports of condemned carcasses and a trace back. Positive herds were confirmed by cross-reference to purified protein derivative tests conducted by the District Veterinary Office. The GPS coordinates were transferred into ArcView 3.2 and laid on the map of Namwala District alongside physical features, including national parks, game management areas, and flood plains. Questionnaires were administered across 96 independent households to assess risk factors of bTB transmission., Results: The results revealed a "clustered" spatial distribution of the disease in cattle in Namwala District of Zambia, particularly significant in the eastern interface areas of the district (p=0.006 using Moran's I). Abattoir to production area trace back revealed a herd-level prevalence of 36.4% (95% CI=26.7-46.3%) among cattle herds in Namwala District, whereas individual animal prevalence ranged from 0% to 14% (95% CI=2.4-26.2%). Further, GPS data indicated that the majority of the positive herds were located at the livestock/wildlife interface area. Contacts with wildlife, coupled with sharing grazing, and watering points were found to be significant risk factors for bTB transmission., Conclusion: This study demonstrated the presence of bTB in cattle and associated spatial risk factors. In particular, bTB was observed to be a function of animal location within the livestock/wildlife interface area. GIS is thus an applicable and important tool in studying disease distribution., (Copyright: © Tembo, et al.)