Late Devonian aquatic environments hosted the fin-to-limb transition in vertebrates. Upper Devonian (ca. 365−360 Ma) strata in Pennsylvania, USA, preserve a diversity of fishes and tetrapods in coastal marine to fluvial depositional environments, making this region ideal for investigating the ecology and evolution of Late Devonian vertebrates. A key unresolved issue has been reconstructing the specific aquatic habitats that hosted various vertebrates during this period. Specifically, the salinity of environments spanning fresh to shallow marine water is difficult to discern from sedimentological and paleontological analyses alone. Here, we analyze rare earth elements and yttrium (REY) as well as stable oxygen and sulfur isotope compositions (δ18O, δ34S) in fossil vertebrate bioapatite from late Famennian (ca. 362−360 Ma) strata of the Catskill and Lock Haven formations in the Appalachian Basin, USA, to determine the relative salinity of their aquatic environments. These results confirm the ecological euryhalinity of several taxa (Bothriolepis sp., tristichopterids, and Holoptychius sp.). Our results are the first demonstrating that some early tetrapod species occupied unequivocally freshwater habitats by late Famennian time (ca. 362−360 Ma). Our study shows that integrating sedimentological and paleontological data with combined oxygen and sulfur isotope analysis allows precise tracing of the relative salinity of vertebrate habitats deep in the past.