Developing countries such as Malawi require improved access to isotope tracer tools to better characterize and manage water resources threatened by land development, deforestation and climate change. This is the first published study to use an isotope facility developed in Malawi for this purpose, instead of relying upon sample analyses from abroad. Results from this new facility are used to evaluate an important Lake Malawi catchment in the Rift Valley. This work successfully established a stable-isotope baseline, hydrochemical signatures, and system conceptualization against which future policy change and management strategies may be measured. Precipitation isotopic composition was consistent with the Global Meteoric Water Line, but varied, confirming different precipitation systems nationally. Groundwater largely followed a Local Meteoric Water Line, with limited isotopic variation indicating predominant areal groundwater recharge, but with dry-season evaporative enrichment of groundwater near Lake Malawi. Surface-water isotopes widely varied with local precipitation, suggesting the latter accounted for wet-season river flows, but upstream dambo (complex wetlands occupying a shallow, seasonal waterlogged depression) helped sustain dry-season flows. Isotope capacity reinforced water-resource conceptualization and provenance in a hydrologically complex, but not atypical, Rift Valley system, exhibiting a noted complexity of groundwater–surface-water interactions. The latter, critical to integrated water resource management, requires more focused study, to which an expanded array of isotopes will contribute to tracking Sustainable Development Goal 6 targets. This study and future catchment studies should help underpin Malawian water-resource policy implementation on several identified fronts.