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In Uganda, the borders are highly porous to animal movement, which may contribute to zoonotic disease spread. We piloted an animal adaptation of an existing human‐focused toolkit to collect data on animal movement patterns and interactions to inform One Health programs. During January 2020, we conducted focus group discussions and key informant interviews with participatory mapping of 2 national‐level One Health stakeholders and 2 local‐level abattoir representatives from Kampala. Zoonotic disease hotspots changed in 2020 compared with reports from 2017–2019. In contrast to local‐level participants, national‐level participants highlighted districts rather than specific locations. Everyone discussed livestock species; only national‐level participants mentioned wildlife. Participants described seasonality differently. Stakeholders used the results to identify locations for zoonotic disease interventions and sites for future data collection. This implementation of an animal‐adapted population mobility mapping exercise highlights the importance of multisectoral initiatives to promote One Health border health approaches. [ABSTRACT FROM AUTHOR]