Freshwater systems from the Yungas Forest to the semiarid Chaco Plain (Argentina, Bolivia y Paraguay, South America) connect critical environments for migratory fishes threatened by anthropogenic pressures. We examined habitat use of dorado (Salminus brasiliensis) and s & aacute;balo (Prochilodus lineatus) across ontogeny using Sr-87/Sr-86 ratios. An isotopic baseline was established by sampling water at eight sites along an altitudinal gradient (900-200 m a.s.l.), and fish were collected from headwaters (Yungas) and lower sections (Chaco Plain) of the R & iacute;o del Valle-Bermejito (RDV) system (Argentina). A positive Sr-87/Sr-86-altitude gradient and seasonal variability were detected in the RDV basin. Otolith Sr-87/Sr-86 transects suggested that most dorado and s & aacute;balo from the Popay & aacute;n River in El Rey National Park (> 97%) followed a migratory pattern consistent with natal origins in the Chaco Plain, migrating upstream in early years and remaining in headwaters thereafter. We recorded the oldest s & aacute;balo to date (33 years), extending species longevity by 8 years and emphasizing the conservation value of the Popay & aacute;n population. In contrast, Chaco Plain fish exhibited higher plasticity, with five migration patterns suggesting multiple natal sites and extensive connectivity across main rivers (e.g., Bermejo) and temporary wetlands (e.g., Quirquincho). These findings underscore the role of headwaters in sustaining long-lived breeders and highlight Chaco wetlands as key nurseries during early life. Conservation and fisheries management should therefore adopt a holistic, basin-wide approach that integrates protection of headwaters, nursery areas, and migratory corridors.