The Beaufort Group of the Main Karoo Basin of South Africa provides an uninterrupted record of fluvio-lacustrine sedimentation from the Middle Permian to the Middle Triassic and covers an area of approximately 200,000 km², about 20% of the country's surface area. A diverse range of vertebrate taxa including fish, therapsids, amphibians, archosauromorphs and parareptiles, has been recovered from this rock succession and provides an ideal sampleset to investigate temporal and geographical variations of local hydrology using stable isotopes. Questions relating to temporal and biological variations in oxygen stable isotope ratios of apatite phosphate (δ¹⁸O_p) in Beaufort tetrapod taxa have previously been addressed, but geographic variation of bioapatite-recorded δ¹⁸O_p across the Permo-Triassic of South Africa has not yet been studied. Here we highlight variations in δ¹⁸O_p recorded over a large geographic spread in the Karoo basin for four Permo-Triassic vertebrate assemblage zones (AZ). Tetrapods, mainly therapsids, were sampled from two Permian (Cistecephalus AZ and Daptocephalus AZ) and two Triassic biozones (Lystrosaurus declivis AZ and Cynognathus AZ). For each of these biozones, fossils were sampled from several localities and their δ¹⁸O_p values compared. Results from the Permian data showed no isotopic difference in apatite from localities divided by longitude 24°. Isotopic differences seem to appear further east in the Basin. Most of the Permian localities (restricted to the south-western part of the Basin) were under the influence of a water system originating from the southern mountainous source terrain. Oxygen isotope compositions of vertebrates from the Lystrosaurus declivis AZ show a significant latitudinal gradient interpreted as an evaporation effect from a southern source in the mountains running toward the north. The Basin during the Cynognathus AZ was under a similar pattern with an evaporation effect highlighted with lower δ¹⁸O values in the south and higher in the north.