Understanding habitat use patterns and life history movements of estuarine and freshwater fish are key to the conservation of native fish populations, particularly migratory (e.g. diadromous, potadromous) species or those that use estuaries as nursery areas. Here, we aimed to evaluate the use of otolith 87Sr/86Sr isotopic ratios to reconstruct estuarine and freshwater habitat use patterns of both native and invasive fish species in several South Australian estuaries. Specifically, we analysed the variation in water 87Sr/86Sr isotopic composition throughout estuarine habitats and freshwater stream end-members in the Onkaparinga, Hindmarsh, and Inman estuaries, as well as the Coorong, Lower Lakes and Murray Mouth; and compared it to the corresponding values in the edge of the otoliths of fish collected at the same sites. In addition, profile analyses of otolith 87Sr/86Sr covering entire fish life histories were performed to characterise potential migratory and habitat use patterns. Overall, 87Sr/86Sr mixing curves varied among estuaries, and the potential to effectively use these natural tags to examine habitat use and individual fish movements is discussed, with results linked to the geological landscape and physical features of these estuarine systems. Ultimately, mapping natural 87Sr/86Sr variations and developing baseline isoscapes for otolith geochemical studies will provide an enhanced framework to unravel fish habitat use and connectivity patterns.