This Transboundary Diagnostic Analysis (TDA) was conducted to provide information for strategic action and an investment program addressing key environmental issues, poverty alleviation and sustainable development in Lake Victoria Basin. The study was based on primary survey data and empirical secondary information analyzed using the Global International Waters Assessment (GIWA) model and results validated by stakeholders at all levels. A number of transboundary major perceived problems and issues and their impacts are evaluated, focusing on root causes, specific sources, locations and sectors. They include; Decline in endemic and commercial fish stocks, threats to biodiversity, declines in aquatic environmental quality, climatic change and variability, degradation of catchments landscapes and habitats, invasive and introduced species, deteriorating infrastructure and amenities, decline in human health and conflicts in resource use. This TDA is distinct as the first and most comprehensive analysis of water-related issues on the Lake Victoria basin, which provides a benchmark for future monitoring of the health of the basin ecosystem.