Heterocotyle tokoloshei sp. nov. is described from the gills of a single Short-tail stingray, Dasyatis brevicaudata, kept in captivity at the Two Oceans Aquarium in Cape Town, South Africa. The stingray exhibited laboured gill ventilation and deteriorating health on exhibit and was removed to the quarantine area for parasitological study and treatment. A 12 h bath treatment of praziquantel at 20mg/l, pre-dissolved in ethanol, removed 3084 parasites from the gills of the ray. However, the presence of a large number of eggs 24 h post-treatment indicated that viable egg laying adults remained on the gills and that the treatment was not 100% effective. Praziquantel was subsequently administered orally by intubation to the same ray at 150 mg/kg under anaesthetic (2-phenoxyethanol at 0.15 ml/l for approximately 1 h), which resulted in the removal of approximately 392 000 parasites from the gills 12 h post-oral treatment. Twenty-four h post-oral treatment, 3383 worms, but no eggs were recovered. No worms or eggs were recovered 48 h to 10 days post-oral treatment. The ray died approximately 30 days after the completion of the treatment. Heterocotyle tokoloshei sp. nov. is the first Heterocotyle species described from South Africa and represents the first record of a pathogenic Heterocotyle species. The new species can be distinguished from the other 16 species in the genus by the distal region of the male copulatory organ which has distinct small spines and by the morphology of the male copulatory organ accessory piece. Eggs of H. tokoloshei sp. nov. are laid singly and hatch spontaneously between 5 and 8 days at 18°C.