AbstractThe ManguruyúZungaro jahu, endemic to the La Plata Basin, is one of the largest neotropical fish species whose life history is virtually unknown. The objectives of this study were to determine ifZ. jahuis a large‐scale migratory species and to describe habitat use throughout ontogeny using the otolith Sr isotope (87Sr/86Sr) ratio. Surface water samples were collected in 2020 at four sites representing the main water courses of the Great Pantanal in Brazil (upper Paraguay, Cuiabá, São Lourenço and Vermelho rivers). Eighteen fish individuals were collected from the Paraná River (Argentina). New water87Sr/86Sr data were incorporated into previously established statistical models to infer the fish habitat use through the otolith87Sr/86Sr ratios. The water87Sr/86Sr ratio of the upper Paraguay tributaries varied (0.7209 and 0.7229) and were the highest in the La Plata Basin. The dispersal area ofZ. jahuwas restricted to the surroundings of the confluence of the Paraná and Paraguay rivers. Fish showed chemical signatures compatible with those of the middle Paraná (88%), Paraguay‐Pilcomayo‐Bermejo (50%) and the lower/upper Paraná (66%). The discriminant analysis based on otolith core indicated natal origins from the middle Paraná (37%), upper Paraná (32%), and Paraguay sub‐basin (32%). The dispersal area ofZ. jahumigrations could be restricted to the surroundings of the confluence of Paraná and Paraguay, which highlights the need to review regional fishing regulations, especially between latitudes 30°–34° S.