Value chain (VC) support has gained momentum in development and poverty reduction strategies. Achieving food and nutrition security and decent livelihoods within planetary boundaries is impossible without sustainable food VCs. Yet, applicable tools for holistic sustainability assessment along food VCs are lacking. The German BMZ funded the development and testing of a participatory, feasible method, Sustainability Hot Spot Analysis (SHSA) 2.0—under the One World – No Hunger (SEWOH) initiative. SHSA 2.0, a refinement of the Wuppertal Institute’s method, was piloted in Uganda’s Irish potato and Nile perch VCs, aiming at empirical VC studies, policy recommendations, and tool development. Using mixed methods (surveys, interviews, FGDs, PRA, PhotoVoice), sustainability hot spots (SHS) were identified and validated with stakeholders.In the potato VC, 12 SHS were identified, mainly in production and environmental dimensions (soil health, input use, food loss), alongside social (fairness, knowledge co-creation, agency) and economic issues (economic resilience, connectivity, equitable benefits). Though challenges persist, the low environmental footprint and organizational potential represent sustainability entry points. In the Nile perch VC, 14 SHS emerged: biodiversity loss, water quality decline, illegal fishing, poor infrastructure, limited fisher agency, legal gaps, and economic vulnerability. Despite critical ecological impacts, Nile perch supports millions of livelihoods. Enhancing domestic value addition, controlling overfishing, and community-based fishery management are urgent.We conclude that SHSA offers a practical, holistic tool for ex-ante VC evaluation, especially when data is scarce. Sustainability hot spots in Uganda’s potato and Nile perch VCs highlight critical production issues, with policy recommendations including training, contract farming, better storage, and habitat protection.